The amygdala is important for processing emotion, including negative emotion such as anxiety and depression induced by chronic pain. Although remarkable progress has been achieved in recent years on amygdala regulation of both negative (fear) and positive (reward) behavioral responses, our current understanding is still limited regarding how the amygdala processes and integrates these negative and positive emotion responses within the amygdala circuits. In this study with optogenetic stimulation of specific brain circuits, we investigated how amygdala circuits regulate negative and positive emotion behaviors, using pain as an emotional assay in male rats. We report here that activation of the excitatory pathway from the parabrachial nucleus (PBN) that relays peripheral pain signals to the central nucleus of amygdala (CeA) is sufficient to cause behaviors of negative emotion including anxiety, depression, and aversion in normal rats. In strong contrast, activation of the excitatory pathway from basolateral amygdala (BLA) that conveys processed corticolimbic signals to CeA dramatically opposes these behaviors of negative emotion, reducing anxiety and depression, and induces behavior of reward. Surprisingly, activating the PBN-CeA pathway to simulate pain signals does not change pain sensitivity itself, but activating the BLA-CeA pathway inhibits basal and sensitized pain. These findings demonstrate that the pain signal conveyed through the PBN-CeA pathway is sufficient to drive negative emotion and that the corticolimbic signal via the BLA-CeA pathway counteracts the negative emotion, suggesting a top-down brain mechanism for cognitive control of negative emotion under stressful environmental conditions such as pain.SIGNIFICANCE STATEMENT It remains unclear how the amygdala circuits integrate both negative and positive emotional responses and the brain circuits that link peripheral pain to negative emotion are largely unknown. Using optogenetic stimulation, this study shows that the excitatory projection from the parabrachial nucleus to the central nucleus of amygdala (CeA) is sufficient to drive behaviors of negative emotion including anxiety, depression, and aversion in rats. Conversely, activation of the excitatory projection from basolateral amygdala to CeA counteracts each of these behaviors of negative emotion. Thus, this study identifies a brain pathway that mediates pain-driven negative emotion and a brain pathway that counteracts these emotion behaviors in a top-down mechanism for brain control of negative emotion.

Regulator of G protein signaling (RGS) proteins are negative regulators of heterotrimeric G proteins that act by accelerating Gα-mediated GTPase activity to terminate G protein-coupled receptor-associated signaling. RGS8 is expressed in several brain regions involved with movement and mood. To investigate the role of RGS8 in vivo, we generated transgenic mice overexpressing brain RGS8 (RGS8tg). RGS8 gene and protein expressions were examined by real-time PCR and immunohistochemistry, respectively, and a significant increase in RGS8 protein was detected in the hippocampal CA1 region compared with wild-type mice (WT). We characterized the phenotypic traits, and found that RGS8tg showed decreased depressive-like behavior in the forced swimming test (FST). Previously, RGS8 was identified as a potent negative regulator of melanin-concentrating hormone receptor 1 (MCHR1), whose activation is mainly involved in energy homeostasis and emotional processing. Interestingly, acute oral administration of MCHR1 antagonist SNAP94847 did not have antidepressant-like effects on RGS8tg in the FST, but did show antidepressant effects on WT. In contrast, selective noradrenaline reuptake inhibitor desipramine had a significant effect on RGS8tg in the FST. MCHR1 is enriched in a subset of primary cilia, as sensory organelles that mediate extracellular signaling. Immunohistochemical analyses revealed significant elongation of MCHR1-positive cilia in the CA1 region of RGS8tg compared with WT. Taken together, these findings suggest that RGS8 participates in modulation of depression-like behavior through ciliary MCHR1 expressed in the CA1 region. The present study may support the possible modulation of RGS8 function in mood disorders.

Neural stem cells (NSCs) constitute an endogenous reservoir for neurons that could potentially be harnessed for regenerative therapies in disease contexts such as neurodegeneration. However, in Alzheimer's disease (AD), NSCs lose plasticity and thus possible regenerative capacity. We investigate how NSCs lose their plasticity in AD by using starPEG-heparin-based hydrogels to establish a reductionist 3D cell-instructive neuro-microenvironment that promotes the proliferative and neurogenic ability of primary and induced human NSCs. We find that administration of AD-associated Amyloid-β42 causes classical neuropathology and hampers NSC plasticity by inducing kynurenic acid (KYNA) production. Interleukin-4 restores NSC proliferative and neurogenic ability by suppressing the KYNA-producing enzyme Kynurenine aminotransferase (KAT2), which is upregulated in APP/PS1dE9 mouse model of AD and in postmortem human AD brains. Thus, our culture system enables a reductionist investigation of regulation of human NSC plasticity for the identification of potential therapeutic targets for intervention in AD.

In multiple sclerosis patients, demyelination is prominent in both the white and gray matter. Chronic clinical deficits are known to result from acute or chronic injury to the myelin sheath and inadequate remyelination. The underlying molecular mechanisms of remyelination and its failure remain currently unclear. Recent studies have recognized G protein-coupled receptor 17 (GPR17) as an important regulator of oligodendrocyte development and remyelination. So far, the relevance of GPR17 for myelin repair was mainly tested in remyelinating white matter lesions. The relevance of GPR17 for gray matter remyelination as well as remyelination of chronic white matter lesions was not addressed so far. Here, we provide a detailed characterization of GPR17 expression during experimental de- and remyelination. Experimental lesions with robust and limited endogenous remyelination capacity were established by either acute or chronic cuprizone-induced demyelination. Furthermore, remyelinating lesions were induced by the focal injection of lysophosphatidylcholine (LPC) into the corpus callosum. GPR17 expression was analyzed by complementary techniques including immunohistochemistry, in situ hybridization, and real-time PCR. In control animals, GPR17+ cells were evenly distributed in the corpus callosum and cortex and displayed a highly ramified morphology. Virtually all GPR17+ cells also expressed the oligodendrocyte-specific transcription factor OLIG2. After acute cuprizone-induced demyelination, robust endogenous remyelination was evident in the white matter corpus callosum but not in the gray matter cortex. Endogenous callosal remyelination was paralleled by a robust induction of GPR17 expression which was absent in the gray matter cortex. Higher numbers of GPR17+ cells were as well observed after LPC-induced focal white matter demyelination. In contrast, densities of GPR17+ cells were comparable to control animals after chronic cuprizone-induced demyelination indicating quiescence of this cell population. Our findings demonstrate that GPR17 expression induction correlates with acute demyelination and sufficient endogenous remyelination. This strengthens the view that manipulation of this receptor might be a therapeutic opportunity to support endogenous remyelination.

The identification of novel genes underlying complex mouse behavioral traits remains an important step in understanding normal brain function and its dysfunction in mental health disorders. To identify dominant mutations that influence locomotor activity, we performed a mouse N-ethyl-N-nitrosourea (ENU) forward mutagenesis screen and mapped several loci as quantitative traits. Here we describe the fine-mapping and positional cloning of a hyperactivity locus mapped to the medial portion of mouse chromosome four. We employed a modified recombinant progeny testing approach to fine-map the confidence interval from ≈20 Mb down to ≈5 Mb. Whole exome resequencing of all exons in this region revealed a single missense mutation in the adhesion G protein-coupled receptor brain-specific angiogenesis inhibitor 2 (Bai2). This mutation, R619W, is located in a critical extracellular domain that is a hotspot for mutations in this receptor class. We find that in two different mammalian cell lines, surface expression of Bai2 R619W is markedly reduced relative to wild-type Bai2, suggesting that R619W is a loss-of-function mutation. Our results highlight the powerful combination of ENU mutagenesis and next-generation sequencing to identify specific mutations that manifest as subtle behavioral phenotypes.

CD4+ follicular helper T (Tfh) cells have been shown to be critical for the activation of germinal center (GC) B-cell responses. Similar to other infections, Plasmodium infection activates both GC as well as non-GC B cell responses. Here, we sought to explore whether Tfh cells and GC B cells are required to eliminate a Plasmodium infection. A CD4 T cell-targeted deletion of the gene that encodes Bcl6, the master transcription factor for the Tfh program, resulted in complete disruption of the Tfh response to Plasmodium chabaudi in C57BL/6 mice and consequent disruption of GC responses and IgG responses and the inability to eliminate the otherwise self-resolving chronic P. chabaudi infection. On the other hand, and contrary to previous observations in immunization and viral infection models, Signaling Lymphocyte Activation Molecule (SLAM)-Associated Protein (SAP)-deficient mice were able to activate Tfh cells, GC B cells, and IgG responses to the parasite. This study demonstrates the critical role for Tfh cells in controlling this systemic infection, and highlights differences in the signals required to activate GC B cell responses to this complex parasite compared with those of protein immunizations and viral infections. Therefore, these data are highly pertinent for designing malaria vaccines able to activate broadly protective B-cell responses.

The cytoskeletal protein filamin A (FLNA) has been suggested to play an important role in the responsiveness of GH-secreting pituitary tumors to somatostatin receptor subtype 2 (SSTR2) agonists, by regulating SSTR2 expression and signaling. However, the underlying mechanisms are unknown. Here, we use fast multi-color single-molecule microscopy to image individual SSTR2 and FLNA molecules at the surface of living cells with unprecedented spatiotemporal resolution. We find that SSTR2 and FLNA undergo transient interactions, which occur preferentially along actin fibers and contribute to restraining SSTR2 diffusion. Agonist stimulation increases the localization of SSTR2 along actin fibers and, subsequently, SSTR2 clustering and recruitment to clathrin-coated pits (CCPs). Interfering with FLNA-SSTR2 binding with a dominant-negative FLNA fragment increases SSTR2 mobility, hampers the formation and alignment of SSTR2 clusters along actin fibers, and impairs both SSTR2 recruitment to CCPs and SSTR2 internalization. These findings indicate that dynamic SSTR2-FLNA interactions critically control the nanoscale localization of SSTR2 at the plasma membrane and are required for coupling SST2R clustering to internalization. These mechanisms explain the critical role of FLNA in the control of SST2R expression and signaling and suggest the possibility of targeting SSTR2-FLNA interactions for the therapy of pharmacologically resistant GH-secreting pituitary tumors.

Enteroendocrine cells (EEs) are interspersed between enterocytes and stem cells in the Drosophila intestinal epithelium. Like enterocytes, EEs express components of the immune deficiency (IMD) innate immune pathway, which activates transcription of genes encoding antimicrobial peptides. The discovery of large lipid droplets in intestines of IMD pathway mutants prompted us to investigate the role of the IMD pathway in the host metabolic response to its intestinal microbiota. Here we provide evidence that the short-chain fatty acid acetate is a microbial metabolic signal that activates signaling through the enteroendocrine IMD pathway in a PGRP-LC-dependent manner. This, in turn, increases transcription of the gene encoding the endocrine peptide Tachykinin (Tk), which is essential for timely larval development and optimal lipid metabolism and insulin signaling. Our findings suggest innate immune pathways not only provide the first line of defense against infection but also afford the intestinal microbiota control over host development and metabolism.

Investigators have long suspected that pathogenic microbes might contribute to the onset and progression of Alzheimer's disease (AD) although definitive evidence has not been presented. Whether such findings represent a causal contribution, or reflect opportunistic passengers of neurodegeneration, is also difficult to resolve. We constructed multiscale networks of the late-onset AD-associated virome, integrating genomic, transcriptomic, proteomic, and histopathological data across four brain regions from human post-mortem tissue. We observed increased human herpesvirus 6A (HHV-6A) and human herpesvirus 7 (HHV-7) from subjects with AD compared with controls. These results were replicated in two additional, independent and geographically dispersed cohorts. We observed regulatory relationships linking viral abundance and modulators of APP metabolism, including induction of APBB2, APPBP2, BIN1, BACE1, CLU, PICALM, and PSEN1 by HHV-6A. This study elucidates networks linking molecular, clinical, and neuropathological features with viral activity and is consistent with viral activity constituting a general feature of AD.

Human type A Enteroviruses (EV-As) cause diseases ranging from hand-foot-and-mouth disease to poliomyelitis-like disease. Although cellular receptors are identified for some EV-As, they remain elusive for the majority of EV-As. We identify the cell surface molecule KREMEN1 as an entry receptor for coxsackievirus A10 (CV-A10). Whereas loss of KREMEN1 renders cells resistant to CV-A10 infection, KREMEN1 overexpression enhances CV-A10 binding to the cell surface and increases susceptibility to infection, indicating that KREMEN1 is a rate-limiting factor for CV-A10 infection. Furthermore, the extracellular domain of KREMEN1 binds CV-A10 and functions as a neutralizing agent during infection. Kremen-deficient mice are resistant to CV-A10-induced lethal paralysis, emphasizing the relevance of Kremen for infection in vivo. KREMEN1 is also essential for infection by a phylogenetic and pathogenic related group of EV-As. Collectively these findings highlight the importance of KREMEN1 for these emerging pathogens and its potential as an antiviral therapeutic target.

Neurons localize mRNAs near synapses where their translation can be regulated by synaptic demand and activity. Differences in the 3' UTRs of mRNAs can change their localization, stability, and translational regulation. Using 3' end RNA sequencing of microdissected rat brain slices, we discovered a huge diversity in mRNA 3' UTRs, with many transcripts showing enrichment for a particular 3' UTR isoform in either somata or the neuropil. The 3' UTR isoforms of localized transcripts are significantly longer than the 3' UTRs of non-localized transcripts and often code for proteins associated with axons, dendrites, and synapses. Surprisingly, long 3' UTRs add not only new, but also duplicate regulatory elements. The neuropil-enriched 3' UTR isoforms have significantly longer half-lives than somata-enriched isoforms. Finally, the 3' UTR isoforms can be significantly altered by enhanced activity. Most of the 3' UTR plasticity is transcription dependent, but intriguing examples of changes that are consistent with altered stability, trafficking between compartments, or local "remodeling" remain.

White matter injury (WMI) of prematurity is associated with a spectrum of neurological disorders ranging from mild cognitive and behavioral deficits to cerebral palsy. Translational studies have implicated impaired oligodendrocyte development after hypoxia as the primary cause of WMI, but the underlying mechanisms remain poorly understood. The goal of this study was to identify alterations in the expression of oligodendrocyte precursor cell transcription factors in a mouse model of transient mild global hypoxia. Postnatal day (P) 7 mouse pups were exposed to hypoxia (7.5% O2) for 60minutes. We compared oligodendrocyte differentiation and subsequent myelin formation between hypoxia and sham animals at P9, P14 and P28 by examining the expression of key transcription factor regulators of oligodendrocyte differentiation (Ascl1, Olig1, Olig2, and Nkx2.2), as well as APC, a mature oligodendrocyte marker, in the major white matter regions including the corpus callosum, external capsule and anterior commissure. We also examined the effect on myelin formation by examining two myelin specific protein constituents, myelin associated glycoprotein (MAG) and myelin basic protein (MBP), in white matter tracts and whole brain lysate respectively. We found that transient hypoxia at P7 altered the expression of Ascl1, Olig1 and Nkx2.2, resulting in delayed myelination in the external capsule. In addition, our study showed that oligodendrocyte progenitor cells specified several days prior to a hypoxic event are more susceptible to maturation arrest than those specified shortly prior to hypoxia. Our results suggest that alterations of Ascl1, Olig1 and Nkx2.2 underlie impaired oligodendrocyte differentiation and deficient myelination in WMI. These transcription factors are potential therapeutic targets for the treatment of WMI in preterm infants.

Human induced pluripotent stem cells (iPSCs) were generated from exfoliated renal epithelial cells isolated from a urine sample of a 31-year-old healthy woman. Epithelial cells were characterized for the expression of E-cadherin and reprogrammed using non-integrating Sendai viral vectors. The urine-derived iPSC line (designated as MUSIi005-A) was karyotypically normal, expressed pluripotent markers, differentiated into cells of three embryonic germ layers, and showed no viral and transgene expressions at passage 29. Our protocol offers a non-invasive and efficient approach for iPSC generation from patients with genetic or acquired disorders.

Prior to forming and refining synaptic connections, axons of projection neurons navigate long distances to their targets. While much is known about guidance cues for axon navigation through intermediate choice points, whether and how axons are organized within tracts is less clear. Here we analyze the organization of retinal ganglion cell (RGC) axons in the developing mouse retinogeniculate pathway. RGC axons are organized by both eye-specificity and topography in the optic nerve and tract: ipsilateral RGC axons are segregated from contralateral axons and are offset laterally in the tract relative to contralateral axon topographic position. To identify potential cell-autonomous factors contributing to the segregation of ipsilateral and contralateral RGC axons in the visual pathway, we assessed their fasciculation behavior in a retinal explant assay. Ipsilateral RGC neurites self-fasciculate more than contralateral neurites in vitro and maintain this difference in the presence of extrinsic chiasm cues. To further probe the role of axon self-association in circuit formation in vivo, we examined RGC axon organization and fasciculation in an EphB1-/- mutant, in which a subset of ipsilateral RGC axons aberrantly crosses the midline but targets the ipsilateral zone in the dorsal lateral geniculate nucleus on the opposite side. Aberrantly crossing axons retain their association with ipsilateral axons in the contralateral tract, indicating that cohort-specific axon affinity is maintained independently of guidance signals present at the midline. Our results provide a comprehensive assessment of RGC axon organization in the retinogeniculate pathway and suggest that axon self-association contributes to pre-target axon organization.

The inhibitory effects of cancer on T cell metabolism have been well established, but the metabolic impact of immunotherapy on tumor cells is poorly understood. Here, we developed a CD4+ T cell-based adoptive immunotherapy protocol that was curative for mice with implanted colorectal tumors. By conducting metabolic profiling on tumors, we show that adoptive immunotherapy profoundly altered tumor metabolism, resulting in glutathione depletion and accumulation of reactive oxygen species (ROS) in tumor cells. We further demonstrate that T cell-derived tumor necrosis factor alpha (TNF-α) can synergize with chemotherapy to intensify oxidative stress and tumor cell death in an NADPH (nicotinamide adenine dinucleotide phosphate hydrogen) oxidase-dependent manner. Reduction of oxidative stress, by preventing TNF-α-signaling in tumor cells or scavenging ROS, antagonized the therapeutic effects of adoptive immunotherapy. Conversely, provision of pro-oxidants after chemotherapy can partially recapitulate the antitumor effects of T cell transfer. These findings imply that reinforcing tumor oxidative stress represents an important mechanism underlying the efficacy of adoptive immunotherapy.

We report the generation of the human iPSC line LEIi004-A from a patient with late-onset non-syndromic retinitis pigmentosa caused by compound heterozygous mutations in the CLN3 gene. Reprogramming of primary dermal fibroblasts was performed using episomal plasmids containing OCT4, SOX2, KLF4, L-MYC, LIN28, shRNA for p53 and mir302/367 microRNA. To create a coisogenic control line, one CLN3 variant was corrected in the patient-iPSC using CRISPR/Cas9 gene editing to generate the iPSC line LEIi004-A-1.

MERRF syndrome is predominantly caused by A8344G mutation in the mitochondrial DNA (mtDNA), affecting MT-TK gene, which impairs the mitochondrial electron transport chain function. Here, we report the generation of two isogenic induced pluripotent stem cell (iPSC) lines, TVGH-iPSC-MRF-Mlow and TVGH-iPSC-MRF-Mhigh, from the skin fibroblasts of a female MERRF patient harboring mtDNA A8344G mutation by using retrovirus transduction system. Both cell lines share the same genetic background except containing different proportions of mtDNA with the A8344G mutation. Both cell lines exhibited the pluripotency and capacity to differentiate into three germ layers.

Disturbances in the nodes of Ranvier are an early phenomenon in many CNS disorders, including the autoimmune demyelinating disease multiple sclerosis (MS). Using an animal model of optic neuritis, a common early symptom of MS, we have investigated nodal and paranodal compartments in the optic nerve during disease progression. Both nodes and paranodes, as identified by immunohistochemistry against sodium channels (Nav) and Caspr, respectively, were observed to increase in length during the late induction phase of the disease, prior to onset of the demyelination and immune cell infiltration characteristic of optic neuritis. These changes were correlated with both axonal stress and microglial/macrophage activation, and were most apparent in the vicinity of the retrobulbar optic nerve head, the unmyelinated region of the optic nerve where retinal ganglion cell axons exit the retina. Using intravitreal glutamate injection as a model of a primary retinal insult, we demonstrate that this can induce similar nodal and paranodal changes. This may suggest that onset of neurodegeneration in the absence of demyelination, as reported in several studies into the nonaffected eyes of MS patients, may give rise to subtle disturbances in the axo-glial junction.

Most eukaryotic parasites are obligately heteroxenous, requiring sequential infection of different host species in order to survive. Toxoplasma gondii is a rare exception to this rule, having a uniquely facultative heteroxenous life cycle. To understand the origins of this phenomenon, we compared development and stress responses in T. gondii to those of its its obligately heteroxenous relative, Hammondia hammondi and have identified multiple H. hammondi growth states that are distinct from those in T. gondii. Of these, the most dramatic difference was that H. hammondi was refractory to stressors that robustly induce cyst formation in T. gondii, and this was reflected most dramatically in its unchanging transcriptome after stress exposure. We also found that H. hammondi could be propagated in vitro for up to 8 days post-excystation, and we exploited this to generate the first ever transgenic H. hammondi line. Overall our data show that H. hammondi zoites grow as stringently regulated, unique life stages that are distinct from T. gondii tachyzoites, and implicate stress sensitivity as a potential developmental innovation that increased the flexibility of the T. gondii life cycle.

Filial imprinting is the behavior observed in chicks during the sensitive or critical period of the first 2-3 days after hatching; however, after this period they cannot be imprinted when raised in darkness. Our previous study showed that temporal augmentation of the endogenous thyroid hormone 3,5,3'-triiodothyronine (T3) in the telencephalon, by imprinting training, starts the sensitive period just after hatching. Intravenous injection of T3 enables imprinting of chicks on days 4 or 6 post-hatching, even when the sensitive period has ended. However, the molecular mechanism of how T3 acts as a determinant of the sensitive period is unknown. Here, we show that Wnt-2b mRNA level is increased in the T3-injected telencephalon of 4-day old chicks. Pharmacological inhibition of Wnt signaling in the intermediate hyperpallium apicale (IMHA), which is the caudal area of the telencephalon, blocked the recovery of the sensitive period following T3 injection. In addition, injection of recombinant Wnt-2b protein into the IMHA helped chicks recover the sensitive period without the injection of T3. Lastly, we showed Wnt signaling to be involved in imprinting via the IMHA region on day 1 during the sensitive period. These results indicate that Wnt signaling plays a critical role in the opening of the sensitive period downstream of T3.

Plasmodium infection begins with the bite of an anopheline mosquito, when sporozoites along with saliva are injected into a vertebrate host. The role of the host responses to mosquito saliva components in malaria remains unclear. We observed that antisera against Anopheles gambiae salivary glands partially protected mice from mosquito-borne Plasmodium infection. Specifically, antibodies to A. gambiae TRIO (AgTRIO), a mosquito salivary gland antigen, contributed to the protection. Mice administered AgTRIO antiserum showed lower Plasmodium liver burden and decreased parasitemia when exposed to infected mosquitoes. Active immunization with AgTRIO was also partially protective against Plasmodium berghei infection. A combination of AgTRIO antiserum and antibodies against Plasmodium circumsporozoite protein, a vaccine candidate, further decreased P. berghei infection. In humanized mice, AgTRIO antiserum afforded some protection against mosquito-transmitted Plasmodium falciparum. AgTRIO antiserum reduced the movement of sporozoites in the murine dermis. AgTRIO may serve as an arthropod-based target against Plasmodium to combat malaria.

We generated human induced pluripotent stem cells (hiPSCs) from dermal fibroblasts of a 51years old female patient homozygous for the mutation c.535 G>A p.G179S on the KCNQ1 gene, causing a severe form of autosomal recessive Long QT Syndrome type 1 (AR-LQT1), not associated with deafness. The hiPSCs, generated using four retroviruses each encoding for a reprogramming factor OCT4, SOX2, KLF4, cMYC, are pluripotent and can differentiate into spontaneously beating cardiomyocytes (hiPSC-CMs).

Astrocytes are complex bushy cells that serve important functions through close contacts between their processes and synapses. However, the spatial interactions and dynamics of astrocyte processes relative to synapses have proven problematic to study in adult living brain tissue. Here, we report a genetically targeted neuron-astrocyte proximity assay (NAPA) to measure astrocyte-synapse spatial interactions within intact brain preparations and at synaptic distance scales. The method exploits resonance energy transfer between extracellularly displayed fluorescent proteins targeted to synapses and astrocyte processes. We validated the method in the striatal microcircuitry following in vivo expression. We determined the proximity of striatal astrocyte processes to distinct neuronal input pathways, to D1 and D2 medium spiny neuron synapses, and we evaluated how astrocyte-to-excitatory synapse proximity changed following cortical afferent stimulation, during ischemia and in a model of Huntington's disease. NAPA provides a simple approach to measure astrocyte-synapse spatial interactions in a variety of experimental scenarios. VIDEO ABSTRACT.

BACKGROUND: Fear conditioning is a form of learning essential for animal survival and used as a behavioral paradigm to study the mechanisms of learning and memory. In mammals, the amygdala plays a crucial role in fear conditioning. In teleost, the medial zone of the dorsal telencephalon (Dm) has been postulated to be a homolog of the mammalian amygdala by anatomical and ablation studies, showing a role in conditioned avoidance response. However, the neuronal populations required for a conditioned avoidance response via the Dm have not been functionally or genetically defined. RESULTS: We aimed to identify the neuronal population essential for fear conditioning through a genetic approach in zebrafish. First, we performed large-scale gene trap and enhancer trap screens, and created transgenic fish lines that expressed Gal4FF, an engineered version of the Gal4 transcription activator, in specific regions in the brain. We then crossed these Gal4FF-expressing fish with the effector line carrying the botulinum neurotoxin gene downstream of the Gal4 binding sequence UAS, and analyzed the double transgenic fish for active avoidance fear conditioning. We identified 16 transgenic lines with Gal4FF expression in various brain areas showing reduced performance in avoidance responses. Two of them had Gal4 expression in populations of neurons located in subregions of the Dm, which we named 120A-Dm neurons. Inhibition of the 120A-Dm neurons also caused reduced performance in Pavlovian fear conditioning. The 120A-Dm neurons were mostly glutamatergic and had projections to other brain regions, including the hypothalamus and ventral telencephalon. CONCLUSIONS: Herein, we identified a subpopulation of neurons in the zebrafish Dm essential for fear conditioning. We propose that these are functional equivalents of neurons in the mammalian pallial amygdala, mediating the conditioned stimulus-unconditioned stimulus association. Thus, the study establishes a basis for understanding the evolutionary conservation and diversification of functional neural circuits mediating fear conditioning in vertebrates.

Dysfunction of the neuronal RNA binding protein RBFOX1 has been linked to epilepsy and autism spectrum disorders. Rbfox1 loss in mice leads to neuronal hyper-excitability and seizures, but the physiological basis for this is unknown. We identify the vSNARE protein Vamp1 as a major Rbfox1 target. Vamp1 is strongly downregulated in Rbfox1 Nes-cKO mice due to loss of 3' UTR binding by RBFOX1. Cytoplasmic Rbfox1 stimulates Vamp1 expression in part by blocking microRNA-9. We find that Vamp1 is specifically expressed in inhibitory neurons, and that both Vamp1 knockdown and Rbfox1 loss lead to decreased inhibitory synaptic transmission and E/I imbalance. Re-expression of Vamp1 selectively within interneurons rescues the electrophysiological changes in the Rbfox1 cKO, indicating that Vamp1 loss is a major contributor to the Rbfox1 Nes-cKO phenotype. The regulation of interneuron-specific Vamp1 by Rbfox1 provides a paradigm for broadly expressed RNA-binding proteins performing specialized functions in defined neuronal subtypes.

Germ cells develop in a microenvironment created by the somatic cells of the gonad [1-3]. Although in males, the germ and somatic support cells lie in direct contact, in females, a thick extracellular coat surrounds the oocyte, physically separating it from the somatic follicle cells [4]. To bypass this barrier to communication, narrow cytoplasmic extensions of the follicle cells traverse the extracellular coat to reach the oocyte plasma membrane [5-9]. These delicate structures provide the sole platform for the contact-mediated communication between the oocyte and its follicular environment that is indispensable for production of a fertilizable egg [8, 10-15]. Identifying the mechanisms underlying their formation should uncover conserved regulators of fertility. We show here in mice that these structures, termed transzonal projections (TZPs), are specialized filopodia whose number amplifies enormously as oocytes grow, enabling increased germ-soma communication. By creating chimeric complexes of genetically tagged oocytes and follicle cells, we demonstrate that follicle cells elaborate new TZPs that push through the extracellular coat to reach the oocyte surface. We further show that growth-differentiation factor 9, produced by the oocyte, drives the formation of new TZPs, uncovering a key yet unanticipated role for the germ cell in building these essential bridges of communication. Moreover, TZP number and germline-soma communication are strikingly reduced in reproductively aged females. Thus, the growing oocyte locally remodels follicular architecture to ensure that its developmental needs are met, and an inability of somatic follicle cells to respond appropriately to oocyte-derived cues may contribute to human infertility.

The intestinal epithelium maintains a remarkable balance between proliferation and differentiation despite rapid cellular turnover. A central challenge is to elucidate mechanisms required for robust control of tissue renewal. Opposing WNT and BMP signaling is essential in establishing epithelial homeostasis. However, it has been difficult to disentangle contributions from multiple sources of morphogen signals in the tissue. Here, to dissect epithelial-autonomous morphogenic signaling circuits, we developed an enteroid monolayer culture system that recapitulates four key properties of the intestinal epithelium, namely the ability to maintain proliferative and differentiated zones, self-renew, polarize, and generate major intestinal cell types. We systematically perturb intrinsic and extrinsic sources of WNT and BMP signals to reveal a core morphogenic circuit that controls proliferation, tissue organization, and cell fate. Our work demonstrates the ability of intestinal epithelium, even in the absence of 3D tissue architecture, to control its own growth and organization through morphogen-mediated feedback.

Heterochromatin-dependent gene silencing is central to the adaptation and survival of Plasmodium falciparum malaria parasites, allowing clonally variant gene expression during blood infection in humans. By assessing genome-wide heterochromatin protein 1 (HP1) occupancy, we present a comprehensive analysis of heterochromatin landscapes across different Plasmodium species, strains, and life cycle stages. Common targets of epigenetic silencing include fast-evolving multi-gene families encoding surface antigens and a small set of conserved HP1-associated genes with regulatory potential. Many P. falciparum heterochromatic genes are marked in a strain-specific manner, increasing the parasite's adaptive capacity. Whereas heterochromatin is strictly maintained during mitotic proliferation of asexual blood stage parasites, substantial heterochromatin reorganization occurs in differentiating gametocytes and appears crucial for the activation of key gametocyte-specific genes and adaptation of erythrocyte remodeling machinery. Collectively, these findings provide a catalog of heterochromatic genes and reveal conserved and specialized features of epigenetic control across the genus Plasmodium.

Mutations in the thyroid hormone (TH) transporter monocarboxylate transporter 8 (MCT8) result in severe intellectual and motor disability. At present, no effective therapy is available to restore TH signaling in MCT8-dependent tissues. Recent in vitro studies in stable overexpression cell models suggested that the function of certain mutant MCT8 proteins, specifically those that affect protein stability and intracellular trafficking (e.g., p.F501del), could be partially recovered by chemical chaperones. However, the effects of chaperones have not been demonstrated in other commonly used models for MCT8 deficiency, including transient overexpression models and patient-derived fibroblasts. Here, we demonstrate that the chemical chaperone 4-phenylbutyric acid (PBA) similarly potentiates the T3 transport function of wild-type and p.F501del mutant MCT8 in transiently transfected COS-1 cells by increasing MCT8 messenger RNA, total protein, and cell surface expression levels. Although PBA also increased the cell surface expression levels of the p.R445L mutant, no functional improvement was observed, which is in line with the proposed important role of Arg445 in substrate translocation. In contrast, PBA showed only minimal effects in ex vivo studies using control or p.F501del patient-derived fibroblasts. Moreover, the MCT8-specific inhibitor silychristin did not change these minimal effects, suggesting that the underlying mechanism is unrelated to the rescue of functional MCT8. Together, these findings indicate that the potency of chaperones to rescue mutant MCT8 function strongly depends on the cellular model and stress the need for further preclinical studies before clinically available chaperones should be considered as a treatment option in patients with MCT8 deficiency.

Adenomatous polyposis coli (APC) mutations cause Wnt pathway activation in human cancers. Current models for APC action emphasize its role in promoting β-catenin degradation downstream of Wnt receptors. Unexpectedly, we find that blocking Wnt receptor activity in APC-deficient cells inhibits Wnt signaling independently of Wnt ligand. We also show that inducible loss of APC is rapidly followed by Wnt receptor activation and increased β-catenin levels. In contrast, APC2 loss does not promote receptor activation. We show that APC exists in a complex with clathrin and that Wnt pathway activation in APC-deficient cells requires clathrin-mediated endocytosis. Finally, we demonstrate conservation of this mechanism in Drosophila intestinal stem cells. We propose a model in which APC and APC2 function to promote β-catenin degradation, and APC also acts as a molecular "gatekeeper" to block receptor activation via the clathrin pathway.

Involution returns the lactating mammary gland to a quiescent state after weaning. The mechanism of involution involves collapse of the mammary epithelial cell compartment. To test whether the cJUN NH2-terminal kinase (JNK) signal transduction pathway contributes to involution, we established mice with JNK deficiency in the mammary epithelium. We found that JNK is required for efficient involution. JNK deficiency did not alter the STAT3/5 or SMAD2/3 signaling pathways that have been previously implicated in this process. Nevertheless, JNK promotes the expression of genes that drive involution, including matrix metalloproteases, cathepsins, and BH3-only proteins. These data demonstrate that JNK has a key role in mammary gland involution post lactation.

Trichogramma evanescens parasitic wasps show large phenotypic plasticity in brain and body size, resulting in a 5-fold difference in brain volume among genetically identical sister wasps. Brain volume scales linearly with body volume in these wasps. This isometric brain scaling forms an exception to Haller's rule, which states that small animals have relatively larger brains than large animals. The large plasticity in brain size may be facilitated by plasticity in neuron size, in the number of neurons, or both. Here, we investigated whether brain isometry requires plasticity in the number and size of monoaminergic neurons that express serotonin (5HT), octopamine (OA), and dopamine (DA). Genetically identical small and large T. evanescens appear to have the same number of 5HT-, OA-, and DA-like immunoreactive cell bodies in their brains, but these cell bodies differ in diameter. This indicates that brain isometry can be facilitated by plasticity in the size of monoaminergic neurons, rather than plasticity in numbers of monoaminergic neurons. Selection pressures on body miniaturization may have resulted in the evolution of miniaturized neural pathways that allow even the smallest wasps to find suitable hosts. Plasticity in the size of neural components may be among the mechanisms that underlie isometric brain scaling while maintaining cognitive abilities in the smallest individuals.

The structural and functional integrity of subgenual cingulate area 25 (A25) is crucial for emotional expression and equilibrium. A25 has a key role in affective networks, and its disruption has been linked to mood disorders, but its cortical connections have yet to be systematically or fully studied. Using neural tracers in rhesus monkeys, we found that A25 was densely connected with other ventromedial and posterior orbitofrontal areas associated with emotions and homeostasis. A moderate pathway linked A25 with frontopolar area 10, an area associated with complex cognition, which may regulate emotions and dampen negative affect. Beyond the frontal lobe, A25 was connected with auditory association areas and memory-related medial temporal cortices, and with the interoceptive-related anterior insula. A25 mostly targeted the superficial cortical layers of other areas, where broadly dispersed terminations comingled with modulatory inhibitory or disinhibitory microsystems, suggesting a dominant excitatory effect. The architecture and connections suggest that A25 is the consummate feedback system in the PFC. Conversely, in the entorhinal cortex, A25 pathways terminated in the middle-deep layers amid a strong local inhibitory microenvironment, suggesting gating of hippocampal output to other cortices and memory storage. The graded cortical architecture and associated laminar patterns of connections suggest how areas, layers, and functionally distinct classes of inhibitory neurons can be recruited dynamically to meet task demands. The complement of cortical connections of A25 with areas associated with memory, emotion, and somatic homeostasis provide the circuit basis to understand its vulnerability in psychiatric and neurologic disorders.SIGNIFICANCE STATEMENT Integrity of the prefrontal subgenual cingulate cortex is crucial for healthy emotional function. Subgenual area 25 (A25) is mostly linked with other prefrontal areas associated with emotion in a dense network positioned to recruit large fields of cortex. In healthy states, A25 is associated with internal states, autonomic function, and transient negative affect. Constant hyperactivity in A25 is a biomarker for depression in humans and may trigger extensive activation in its dominant connections with areas associated with emotions and internal balance. A pathway between A25 and frontopolar area 10 may provide a critical link to regulate emotions and dampen persistent negative affect, which may be explored for therapeutic intervention in depression.

It is now clear that microglia and macrophages are present in brain tumors, but whether or how they affect initiation and development of tumors is not known. Exploiting the advantages of the zebrafish (Danio rerio) model, we showed that macrophages and microglia respond immediately upon oncogene activation in the brain. Overexpression of human AKT1 within neural cells of larval zebrafish led to a significant increase in the macrophage and microglia populations. By using a combination of transgenic and mutant zebrafish lines, we showed that this increase was caused by the infiltration of peripheral macrophages into the brain mediated via Sdf1b-Cxcr4b signaling. Intriguingly, confocal live imaging reveals highly dynamic interactions between macrophages/microglia and pre-neoplastic cells, which do not result in phagocytosis of pre-neoplastic cells. Finally, depletion of macrophages and microglia resulted in a significant reduction of oncogenic cell proliferation. Thus, macrophages and microglia show tumor promoting functions already during the earliest stages of the developing tumor microenvironment.

Membrane vesicles delivered to the cell-division plane fuse with one another to form the partitioning membrane during plant cytokinesis, starting in the cell center. In Arabidopsis, this requires SNARE complexes involving the cytokinesis-specific Qa-SNARE KNOLLE. However, cytokinesis still occurs in knolle mutant embryos, suggesting contributions from KNOLLE-independent SNARE complexes. Here we show that Qa-SNARE SYP132, having counterparts in lower plants, functionally overlaps with the flowering plant-specific KNOLLE. SYP132 mutation causes cytokinesis defects, knolle syp132 double mutants consist of only one or a few multi-nucleate cells, and SYP132 has the same SNARE partners as KNOLLE. SYP132 and KNOLLE also have non-overlapping functions in secretion and in cellularization of the embryo-nourishing endosperm resulting from double fertilization unique to flowering plants. Evolutionarily ancient non-specialized SNARE complexes originating in algae were thus amended by the appearance of cytokinesis-specific SNARE complexes, meeting the high demand for membrane-fusion capacity during endosperm cellularization in angiosperms.

The mechanisms by which feeding and fasting drive rhythmic gene expression for physiological adaptation to daily rhythm in nutrient availability are not well understood. Here we show that, upon feeding, the RNA-binding protein NONO accumulates within speckle-like structures in liver cell nuclei. Combining RNA-immunoprecipitation and sequencing (RIP-seq), we find that an increased number of RNAs are bound by NONO after feeding. We further show that NONO binds and regulates the rhythmicity of genes involved in nutrient metabolism post-transcriptionally. Finally, we show that disrupted rhythmicity of NONO target genes has profound metabolic impact. Indeed, NONO-deficient mice exhibit impaired glucose tolerance and lower hepatic glycogen and lipids. Accordingly, these mice shift from glucose storage to fat oxidation, and therefore remain lean throughout adulthood. In conclusion, our study demonstrates that NONO post-transcriptionally coordinates circadian mRNA expression of metabolic genes with the feeding/fasting cycle, thereby playing a critical role in energy homeostasis.

X-linked Dystonia-Parkinsonism (XDP) is a Mendelian neurodegenerative disease that is endemic to the Philippines and is associated with a founder haplotype. We integrated multiple genome and transcriptome assembly technologies to narrow the causal mutation to the TAF1 locus, which included a SINE-VNTR-Alu (SVA) retrotransposition into intron 32 of the gene. Transcriptome analyses identified decreased expression of the canonical cTAF1 transcript among XDP probands, and de novo assembly across multiple pluripotent stem-cell-derived neuronal lineages discovered aberrant TAF1 transcription that involved alternative splicing and intron retention (IR) in proximity to the SVA that was anti-correlated with overall TAF1 expression. CRISPR/Cas9 excision of the SVA rescued this XDP-specific transcriptional signature and normalized TAF1 expression in probands. These data suggest an SVA-mediated aberrant transcriptional mechanism associated with XDP and may provide a roadmap for layered technologies and integrated assembly-based analyses for other unsolved Mendelian disorders.

In the developing hypothalamus, the fat-derived hormone leptin stimulates the growth of axons from the arcuate nucleus of the hypothalamus (ARH) to other regions that control energy balance. These projections are significantly reduced in leptin deficient (Lepob/ob ) mice and this phenotype is largely rescued by neonatal leptin treatments. However, treatment of mature Lepob/ob mice is ineffective, suggesting that the trophic action of leptin is limited to a developmental critical period. To temporally delineate closure of this critical period for leptin-stimulated growth, we treated Lepob/ob mice with exogenous leptin during a variety of discrete time periods, and measured the density of Agouti-Related Peptide (AgRP) containing projections from the ARH to the ventral part of the dorsomedial nucleus of the hypothalamus (DMHv), and to the medial parvocellular part of the paraventricular nucleus (PVHmp). The results indicate that leptin loses its neurotrophic potential at or near postnatal day 28. The duration of leptin exposure appears to be important, with 9- or 11-day treatments found to be more effective than shorter (5-day) treatments. Furthermore, leptin treatment for 9 days or more was sufficient to restore AgRP innervation to both the PVHmp and DMHv in Lepob/ob females, but only to the DMHv in Lepob/ob males. Together, these findings reveal that the trophic actions of leptin are contingent upon timing and duration of leptin exposure, display both target and sex specificity, and that modulation of leptin-dependent circuit formation by each of these factors may carry enduring consequences for feeding behavior, metabolism, and obesity risk.

Stem cells are highly resistant to viral infection compared to their differentiated progeny; however, the mechanism is mysterious. Here, we analyzed gene expression in mammalian stem cells and cells at various stages of differentiation. We find that, conserved across species, stem cells express a subset of genes previously classified as interferon (IFN) stimulated genes (ISGs) but that expression is intrinsic, as stem cells are refractory to interferon. This intrinsic ISG expression varies in a cell-type-specific manner, and many ISGs decrease upon differentiation, at which time cells become IFN responsive, allowing induction of a broad spectrum of ISGs by IFN signaling. Importantly, we show that intrinsically expressed ISGs protect stem cells against viral infection. We demonstrate the in vivo importance of intrinsic ISG expression for protecting stem cells and their differentiation potential during viral infection. These findings have intriguing implications for understanding stem cell biology and the evolution of pathogen resistance.

In the central nervous system, major histocompatibility complex class I (MHCI) molecules are mainly expressed in neurons, and neuronal MHCI have roles in synapse elimination and plasticity. However, the pathophysiological significance of astroglial MHCI remains unclear. We herein demonstrate that MHCI expression is up-regulated in astrocytes in the medial prefrontal cortex (mPFC) following systemic immune activation by an intraperitoneal injection of polyinosinic-polycytidylic acid (polyI:C) or hydrodynamic interferon (IFN)-γ gene delivery in male C57/BL6J mice. In cultured astrocytes, MHCI/H-2D largely co-localized with exosomes. To investigate the role of astroglial MHCI, H-2D, or sH-2D was expressed in the mPFC of male C57/BL6J mice using an adeno-associated virus vector under the control of a glial fibrillary acidic protein promoter. The expression of astroglial MHCI in the mPFC impaired sociability and recognition memory in mice. Regarding neuropathological changes, MHCI expression in astrocytes significantly activated microglial cells, decreased parvalbumin-positive cell numbers, and reduced dendritic spine density in the mPFC. A treatment with GW4869 that impairs exosome synthesis ameliorated these behavioral and neuropathological changes. These results suggest that the overexpression of MHCI in astrocytes affects microglial proliferation as well as neuronal numbers and spine densities, thereby leading to social and cognitive deficits in mice, possibly via exosomes created by astrocytes.

Centrosomes [1, 2] play a central role during spindle assembly in most animal cells [3]. In early mitosis, they organize two symmetrical microtubule arrays that upon separation define the two poles of the forming spindle. Centrosome separation is tightly regulated [4, 5], occurring through partially redundant mechanisms that rely on the action of microtubule-based dynein and kinesin motors and the actomyosin system [6]. While centrosomes can separate in prophase or in prometaphase after nuclear envelope breakdown (NEBD), prophase centrosome separation optimizes spindle assembly and minimizes the occurrence of abnormal chromosome attachments that could end in aneuploidy [7, 8]. Prophase centrosome separation relies on the activity of Eg5/KIF11, a mitotic kinesin [9] that accumulates around centrosomes in early mitosis under the control of CDK1 and the Nek9/Nek6/7 kinase module [10-17]. Here, we show that Eg5 localization and centrosome separation in prophase depend on the nuclear microtubule-associated protein TPX2 [18], a pool of which localizes to the centrosomes before NEBD. This localization involves RHAMM/HMMR [19] and the kinase Nek9 [20], which phosphorylates TPX2 nuclear localization signal (NLS) preventing its interaction with importin and nuclear import. The pool of centrosomal TPX2 in prophase has a critical role for both microtubule aster organization and Eg5 localization, and thereby for centrosome separation. Our results uncover an unsuspected role for TPX2 before NEBD and define a novel regulatory mechanism for centrosome separation in prophase. They furthermore suggest NLS phosphorylation as a novel regulatory mechanism for spindle assembly factors controlled by the importin/Ran system.

Color vision extracts spectral information by comparing signals from photoreceptors with different visual pigments. Such comparisons are encoded by color-opponent neurons that are excited at one wavelength and inhibited at another. Here, we examine the circuit implementation of color-opponent processing in the Drosophila visual system by combining two-photon calcium imaging with genetic dissection of visual circuits. We report that color-opponent processing of UVshort/blue and UVlong/green is already implemented in R7/R8 inner photoreceptor terminals of "pale" and "yellow" ommatidia, respectively. R7 and R8 photoreceptors of the same type of ommatidia mutually inhibit each other directly via HisCl1 histamine receptors and receive additional feedback inhibition that requires the second histamine receptor Ort. Color-opponent processing at the first visual synapse represents an unexpected commonality between Drosophila and vertebrates; however, the differences in the molecular and cellular implementation suggest that the same principles evolved independently.

Malaria is a devastating illness that causes approximately 500,000 deaths annually. The malaria-causing parasite (Plasmodium genus) uses the process of translational repression to regulate its growth, development, and transmission. As poly(A)-binding proteins (PABP) have been identified as critical components of RNA metabolism and translational repression in model eukaryotes and in Plasmodium, we have identified and investigated two PABPs in Plasmodium yoelii, PyPABP1 and PyPABP2. In contrast to most single-celled eukaryotes, Plasmodium closely resembles metazoans and encodes both a nuclear PABP and a cytosolic PABP; here, we provide multiple lines of evidence in support of this observation. The conserved domain architectures of PyPABP1 and PyPABP2 resemble those of yeast and metazoans, while multiple independent binding assays demonstrated their ability to bind very strongly and specifically to poly(A) sequences. Interestingly, we also observed that purified PyPABP1 forms homopolymeric chains despite exhaustive RNase treatment in vitro. Finally, we show by indirect immunofluorescence assays (IFAs) that PyPABP1 and PyPABP2 are cytoplasm- and nucleus-associated PABPs during the blood stages of the life cycle. Surprisingly, however, PyPABP1 was instead observed to also be localized on the surface of transmitted salivary gland sporozoites and to be deposited in trails when parasites glide on a substrate. This is the third RNA-binding protein verified to be found on the sporozoite surface, and the data may point to an unappreciated RNA-centered interface between the host and parasite. IMPORTANCE Malaria remains one of the great global health problems. The parasite that causes malaria (Plasmodium genus) relies upon exquisite control of its transmission between vertebrate hosts and mosquitoes. One crucial way that it does so is by proactively producing mRNAs needed to establish the new infection but by silencing and storing them until they are needed. One key protein in this process of translational repression in model eukaryotes is poly(A)-binding protein (PABP). Here we have shown that Plasmodium yoelii utilizes both a nuclear PABP and a cytosolic PABP, both of which bind specifically to polyadenylated RNA sequences. Moreover, we find that the cytosolic PABP forms chains in vitro, consistent with its appreciated role in coating the poly(A) tails of mRNA. Finally, we have also verified that, surprisingly, the cytosolic PABP is found on the surface of Plasmodium sporozoites. Taking the data together, we propose that Plasmodium utilizes a more metazoan-like strategy for RNA metabolism using specialized PABPs.

Sphingosine 1-phosphate receptors (S1PR) are G protein-coupled and compose a family with five subtypes, S1P1R-S1P5R. The drug Gilenya® (Novartis, Basel, Switzerland) (Fingolimod; FTY720) targets S1PRs and was the first oral therapy for patients with relapsing-remitting multiple sclerosis (MS). The phosphorylated form of FTY720 (pFTY720) binds S1PRs causing initial agonism, then subsequent receptor internalization and functional antagonism. Internalization of S1P1R attenuates sphingosine 1-phosphate (S1P)-mediated egress of lymphocytes from lymph nodes, limiting aberrant immune function in MS. pFTY720 also exerts direct actions on neurons and glial cells which express S1PRs. In this study, we investigated the regulation of pro-inflammatory chemokine release by S1PRs in enriched astrocytes and microglial cultures. Astrocytes and microglia were stimulated with lipopolysaccharide (LPS) and increases in C-X-C motif chemokine 5 (CXCL5), also known as LIX (lipopolysaccharide-induced CXC chemokine) expression were quantified. Results showed that pFTY720 attenuated LPS-induced CXCL5 (LIX) protein release from astrocytes, as did the S1P1R selective agonist, SEW2871. In addition, pFTY720 blocked messenger ribonucleic acid (mRNA) transcription of the chemokines, (i) CXCL5/LIX, (ii) C-X-C motif chemokine 10 (CXCL10) also known as interferon gamma-induced protein 10 (IP10) and (iii) chemokine (C-C motif) ligand 2 (CCL2) also known as monocyte chemoattractant protein 1 (MCP1). Interestingly, inhibition of sphingosine kinase attenuated LPS-induced increases in mRNA levels of all three chemokines, suggesting that LPS-TLR4 (Toll-like receptor 4) signalling may enhance chemokine expression via S1P-S1PR transactivation. Lastly, these observations were not limited to astrocytes since we also found that pFTY720 attenuated LPS-induced release of CXCL5 from microglia. These data highlight a role for S1PR signalling in regulating the levels of chemokines in glial cells and support the notion that pFTY720 efficacy in multiple sclerosis may involve the direct modulation of astrocytes and microglia.

[18F]Fluorodeoxyglucose-PET/CT (18F-FDG-PET/CT) imaging has been invaluable for visualizing metabolically active adipose tissues in humans with potential anti-diabetic and anti-obesity effects. To explore whether mice display human-like fat depots in anatomically comparable regions, we mapped fat depots using glucose or fatty acid imaging tracers, such as 18F-FDG through PET/CT or [123/125I]-β-methyl-p-iodophenyl-pentadecanoic acid with SPECT/CT imaging, to analogous depots in mice. Using this type of image analysis with both probes, we define a large number of additional areas of high metabolic activity corresponding to novel fat pads. Histological and gene expression analyses validate these regions as bona fide fat pads. Our findings indicate that fat depots of rodents show a high degree of topological similarity to those of humans. Studies involving both glucose and lipid tracers indicate differential preferences for these substrates in different depots and also suggest that fatty acid-based visualized approaches may reveal additional brown adipose tissue and beige depots in humans.

Preservation of a balance between synaptic excitation and inhibition is critical for normal brain function. A number of homeostatic cellular mechanisms have been suggested to play a role in maintaining this balance, including long-term plasticity of GABAergic inhibitory synapses. Many previous studies have demonstrated a coupling of postsynaptic spiking with modification of perisomatic inhibition. Here, we demonstrate that activation of NMDA-type glutamate receptors leads to input-specific long-term potentiation of dendritic inhibition mediated by somatostatin-expressing interneurons. This form of plasticity is expressed postsynaptically and requires both CaMKIIα and the β2 subunit of the GABA-A receptor. Importantly, this process may function to preserve dendritic inhibition, as genetic deletion of NMDAR signaling results in a selective weakening of dendritic inhibition. Overall, our results reveal a new mechanism for linking excitatory and inhibitory input in neuronal dendrites and provide novel insight into the homeostatic regulation of synaptic transmission in cortical circuits.

Increased neuron telencephalic differentiation during deep cortical layer formation has been reported in embryos from diabetic mice. Transitory histaminergic neurons within the mesencephalon/rhombencephalon are responsible for fetal histamine synthesis during development, fibers from this system arrives to the frontal and parietal cortex at embryo day (E) 15. Histamine is a neurogenic factor for cortical neural stem cells in vitro through H1 receptor (H1R) which is highly expressed during corticogenesis in rats and mice. Furthermore, in utero administration of an H1R antagonist, chlorpheniramine, decreases the neuron markers microtubuline associated protein 2 (MAP2) and forkhead box protein 2. Interestingly, in the diabetic mouse model of diabetes induced with streptozotocin, an increase in fetal neurogenesis in terms of MAP2 expression in the telencephalon is reported at E11.5. Because of the reported effects on cortical neuron differentiation of maternal diabetes in one hand and of histamine in the other, here the participation of histamine and H1R on the increased dorsal telencephalic neurogenesis was explored. First, the increased neurogenesis in the dorsal telencephalon at E14 in diabetic rats was corroborated by immunohistochemistry and Western blot. Then, changes during corticogenesis in the level of histamine was analyzed by ELISA and in H1R expression by qRT-PCR and Western blot and, finally, we tested H1R participation in the increased dorsal telencephalic neurogenesis by the systemic administration of chlorpheniramine. Our results showed a significant increase of histamine at E14 and in the expression of the receptor at E12. The administration of chlorpheniramine to diabetic rats at E12 prevented the increased expression of βIII-tubulin and MAP2 mRNAs (neuron markers) and partially reverted the increased level of MAP2 protein at E14, concluding that H1R have an important role in the increased neurogenesis within the dorsal telencephalon of embryos from diabetic rats. This study opens new perspective on the participation of HA and H1R receptor in early corticogenesis in health and disease.

An adverse intrauterine environment may induce adult disease in offspring, but the mechanisms are not well understood. It is reported that fresh embryo transfer (ET) in assisted reproductive technology leads to high maternal estradiol (E2), and prenatal high E2 exposure increases the risk of organ disorders in later life. We found that male newborns and children of fresh ET showed elevated fasting insulin and homeostasis model of assessment for insulin resistance index (HOMA-IR) scores. Male mice with high prenatal estradiol exposure (HE) grew heavier than control mice and developed insulin resistance; they also showed increased food intake, with increased orexigenic hypothalamic neuropeptide Y (NPY) expression. The hypothalamic insulin receptor (INSR) was decreased in male HE mice, associated with elevated promoter methylation. Chronic food restriction (FR) in HE mice reversed insulin resistance and rescued hypothalamic INSR expression by correcting the elevated Insr promoter methylation. Our findings suggest that prenatal exposure to high E2 may induce sex-specific metabolic disorders in later life through epigenetic programming of hypothalamic Insr promoter, and dietary intervention may reverse insulin resistance by remodeling its methylation pattern.

BACKGROUND: For many fish species, retinal function changes between life history stages as part of an encoded developmental program. Retinal change is also known to exhibit plasticity because retinal form and function can be influenced by light exposure over the course of development. Aside from studies of gene expression, it remains largely unknown whether retinal plasticity can provide functional responses to short-term changes in environmental light quality. The aim of this study was to determine whether the structure and function of the fish retina can change in response to altered light intensity and spectrum-not over the course of a developmental regime, but over shorter time periods relevant to marine habitat disturbance. RESULTS: The effects of light environment on sensitivity of the retina, as well as on cone photoreceptor distribution were examined in the Atlantic tarpon (Megalops atlanticus) on 2- and 4-month timescales. In a spectral experiment, juvenile M. atlanticus were placed in either 'red' or 'blue' light conditions (with near identical irradiance), and in an intensity experiment, juveniles were placed in either 'bright' or 'dim' light conditions (with near identical spectra). Analysis of the retina by electroretinography and anti-opsin immunofluorescence revealed that relative to fish held in the blue condition, those in the red condition exhibited longer-wavelength peak sensitivity and greater abundance of long-wavelength-sensitive (LWS) cone photoreceptors over time. Following pre-test dark adaption of the retina, fish held in the dim light required less irradiance to produce a standard retinal response than fish held in bright light, developing a greater sensitivity to white light over time. CONCLUSIONS: The results show that structure and function of the M. atlanticus retina can rapidly adjust to changes in environmental light within a given developmental stage, and that such changes are dependent on light quality and the length of exposure. These findings suggest that the fish retina may be resilient to disturbances in environmental light, using retinal plasticity to compensate for changes in light quality over short timescales.

In Drosophila melanogaster larvae, the prime site of external taste reception is the terminal organ (TO). Though investigation on the TO's implications in taste perception has been expanding rapidly, the sensilla of the TO have been essentially unexplored. In this study, we performed a systematic anatomical and molecular analysis of the TO. We precisely define morphological types of TO sensilla taking advantage of volume electron microscopy and 3D image analysis. We corroborate the presence of five external types of sensilla: papilla, pit, spot, knob, and modified papilla. Detailed 3D analysis of their structural organization allowed a finer discrimination into subtypes. We classify three subtypes of papilla and pit sensilla, respectively, and two subtypes of knob sensilla. Further, we determine the repertoire of receptor genes for each sensillum by analyzing GAL4 driver lines of Ir, Gr, Ppk, and Trp receptor genes. We construct a map of the TO, in which the receptor genes are mapped to neurons of individual sensilla. While modified papillum and spot sensilla are not labeled by any GAL4 driver, neurons of the pit, papilla, and knob type are labeled by partially overlapping but different subsets of GAL4 driver lines of the Ir, Gr, and Ppk gene family. The results suggest that pit, papilla and knob sensilla act in contact chemosensation. However, they likely do these employing different stimulus transduction mechanisms to sense the diverse chemicals of their environment.

Genetic and pathologic data suggest that amyloid beta (Aβ), produced by processing of the amyloid precursor protein, is a major initiator of Alzheimer's disease (AD). To gain new insights into Aβ modulation, we sought to harness the power of the coevolution between the neurotropic parasite Toxoplasma gondii and the mammalian brain. Two prior studies attributed Toxoplasma-associated protection against Aβ to increases in anti-inflammatory cytokines (TGF-β and IL-10) and infiltrating phagocytic monocytes. These studies only used one Toxoplasma strain making it difficult to determine if the noted changes were associated with Aβ protection or simply infection. To address this limitation, we infected a third human amyloid precursor protein AD mouse model (J20) with each of the genetically distinct, canonical strains of Toxoplasma (Type I, Type II, or Type III). We then evaluated the central nervous system (CNS) for Aβ deposition, immune cell responses, global cytokine environment, and parasite burden. We found that only Type II infection was protective against Aβ deposition despite both Type II and Type III strains establishing a chronic CNS infection and inflammatory response. Compared with uninfected and Type I-infected mice, both Type II- and Type III-infected mice showed increased numbers of CNS T cells and microglia and elevated pro-inflammatory cytokines, but neither group showed a >2-fold elevation of TGF-β or IL-10. These data suggest that we can now use our identification of protective (Type II) and nonprotective (Type III) Toxoplasma strains to determine what parasite and host factors are linked to decreased Aβ burden rather than simply with infection.

Inflammation within the CNS is a major component of many neurodegenerative diseases. A characteristic feature is the generation of microglia-derived factors that play an essential role in the immune response. IL-1β is a pro-inflammatory cytokine released by activated microglia, able to exacerbate injury at elevated levels. In the presence of caspase-1, pro-IL-1β is cleaved to the mature cytokine following NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome activation. Growing evidence suggests that ceramide plays a critical role in NLRP3 inflammasome assembly, however, the relationship between ceramide and inflammasome activation in microglia remains unknown. Here, we investigated potential mechanistic links between ceramide as a modulator of NLRP3 inflammasome assembly and the resulting secretion of IL-1β using small bioactive enzyme stimulators and inhibitors of ceramide signaling in wild-type and apoptosis-associated speck-like protein containing a CARD knockout (ASC-/- ) primary microglia. To induce the expression of inflammasome components, microglia were primed prior to experiments. Treatment with sodium palmitate (PA) induced de novo ceramide synthesis via modulation of its synthesizing protein serine palmitoyl transferase resulting in increased IL-1β secretion in microglia. Exposure of microglia to the serine palmitoyl transferase-inhibitor l-cycloserine significantly prevented PA-induced IL-1β secretion. Application of the ceramide analogue C2 and the sphingosine-1-phosphate-receptor agonist Fingolimod (FTY720) up-regulated levels of IL-1β and cleaved caspase-1 in wild-type microglia, whereas ASC-/- microglia were unaffected. HPA-12 inhibition of ceramide transport did not affect inflammasome activation. Taken together, our findings reveal a critical role for ceramide as a positive modulator of NLRP3 inflammasome assembly and the resulting release of IL-1β.

Mitochondrial fusion and fission are critical to heart health; genetically interrupting either is rapidly lethal. To understand whether it is loss of, or the imbalance between, fusion and fission that underlies observed cardiac phenotypes, we engineered mice in which Mfn-mediated fusion and Drp1-mediated fission could be concomitantly abolished. Compared to fusion-defective Mfn1/Mfn2 cardiac knockout or fission-defective Drp1 cardiac knockout mice, Mfn1/Mfn2/Drp1 cardiac triple-knockout mice survived longer and manifested a unique pathological form of cardiac hypertrophy. Over time, however, combined abrogation of fission and fusion provoked massive progressive mitochondrial accumulation that severely distorted cardiomyocyte sarcomeric architecture. Mitochondrial biogenesis was not responsible for mitochondrial superabundance, whereas mitophagy was suppressed despite impaired mitochondrial proteostasis. Similar but milder defects were observed in aged hearts. Thus, cardiomyopathies linked to dynamic imbalance between fission and fusion are temporarily mitigated by forced mitochondrial adynamism at the cost of compromising mitochondrial quantity control and accelerating mitochondrial senescence.

Friedreich's ataxia (FRDA), the most common inherited ataxia, is caused by recessive mutations that reduce the levels of frataxin (FXN), a mitochondrial iron binding protein. We developed an inducible mouse model of Fxn deficiency that enabled us to control the onset and progression of disease phenotypes by the modulation of Fxn levels. Systemic knockdown of Fxn in adult mice led to multiple phenotypes paralleling those observed in human patients across multiple organ systems. By reversing knockdown after clinical features appear, we were able to determine to what extent observed phenotypes represent reversible cellular dysfunction. Remarkably, upon restoration of near wild-type FXN levels, we observed significant recovery of function, associated pathology and transcriptomic dysregulation even after substantial motor dysfunction and pathology were observed. This model will be of broad utility in therapeutic development and in refining our understanding of the relative contribution of reversible cellular dysfunction at different stages in disease.

Appropriate excitatory/inhibitory (E/I) balance is essential for normal cortical function and is altered in some psychiatric disorders, including autism spectrum disorders (ASDs). Cell-autonomous molecular mechanisms that control the balance of excitatory and inhibitory synapse function remain poorly understood; no proteins that regulate excitatory and inhibitory synapse strength in a coordinated reciprocal manner have been identified. Using super-resolution imaging, electrophysiology, and molecular manipulations, we show that cadherin-10, encoded by CDH10 within the ASD risk locus 5p14.1, maintains both excitatory and inhibitory synaptic scaffold structure in cultured cortical neurons from rats of both sexes. Cadherin-10 localizes to both excitatory and inhibitory synapses in neocortex, where it is organized into nanoscale puncta that influence the size of their associated PSDs. Knockdown of cadherin-10 reduces excitatory but increases inhibitory synapse size and strength, altering the E/I ratio in cortical neurons. Furthermore, cadherin-10 exhibits differential participation in complexes with PSD-95 and gephyrin, which may underlie its role in maintaining the E/I ratio. Our data provide a new mechanism whereby a protein encoded by a common ASD risk factor controls E/I ratios by regulating excitatory and inhibitory synapses in opposing directions.SIGNIFICANCE STATEMENT The correct balance between excitatory/inhibitory (E/I) is crucial for normal brain function and is altered in psychiatric disorders such as autism. However, the molecular mechanisms that underlie this balance remain elusive. To address this, we studied cadherin-10, an adhesion protein that is genetically linked to autism and understudied at the cellular level. Using a combination of advanced microscopy techniques and electrophysiology, we show that cadherin-10 forms nanoscale puncta at excitatory and inhibitory synapses, maintains excitatory and inhibitory synaptic structure, and is essential for maintaining the correct balance between excitation and inhibition in neuronal dendrites. These findings reveal a new mechanism by which E/I balance is controlled in neurons and may bear relevance to synaptic dysfunction in autism.

Hermissenda crassicornis is a model for studying the molecular and cellular basis for classical conditioning, based on its ability to associate light with vestibular stimulation. We used confocal microscopy to map histamine (HA), FMRF-amide, and γ-aminobutyric acid (GABA) immunoreactivity in the central nervous system (CNS), eyes, optic ganglia and statocysts of the nudibranchs. For HA immunoreactivity, we documented both consistently and variably labeled CNS structures across individuals. We also noted minor differences in GABA immunoreactivity in the CNS compared to previous work on Hermissenda. Contrary to expectations, we found no evidence for GABA inside the visual or vestibular systems. Instead, we found only FMRFamide- and HA immunoreactivity (FMRFamide: 4 optic ganglion cells, 4-5 hair cells; HA: 3 optic ganglion cells, 8 hair cells). Overall, our results can act as basis for comparisons of nervous systems across nudibranchs, and suggest further exploration of intraspecific plasticity versus evolutionary changes in gastropod nervous systems.

The microdomain that orchestrates action potential initiation in neurons is the axon initial segment (AIS). It has long been considered to be a rather homogeneous domain at the very proximal axon hillock with relatively stable length, particularly in cortical pyramidal cells. However, studies in other brain regions paint a different picture. In hippocampal CA1, up to 50% of axons emerge from basal dendrites. Further, in about 30% of thick-tufted layer V pyramidal neurons in rat somatosensory cortex, axons have a dendritic origin. Consequently, the AIS is separated from the soma. Recent in vitro and in vivo studies have shown that cellular excitability is a function of AIS length/position and somatodendritic morphology, undermining a potentially significant impact of AIS heterogeneity for neuronal function. We therefore investigated neocortical axon morphology and AIS composition, hypothesizing that the initial observation of seemingly homogeneous AIS is inadequate and needs to take into account neuronal cell types. Here, we biolistically transfected cortical neurons in organotypic cultures to visualize the entire neuron and classify cell types in combination with immunolabeling against AIS markers. Using confocal microscopy and morphometric analysis, we investigated axon origin, AIS position, length, diameter as well as distance to the soma. We find a substantial AIS heterogeneity in visual cortical neurons, classified into three groups: (I) axons with somatic origin with proximal AIS at the axon hillock; (II) axons with somatic origin with distal AIS, with a discernible gap between the AIS and the soma; and (III) axons with dendritic origin (axon-carrying dendrite cell, AcD cell) and an AIS either starting directly at the axon origin or more distal to that point. Pyramidal cells have significantly longer AIS than interneurons. Interneurons with vertical columnar axonal projections have significantly more distal AIS locations than all other cells with their prevailing phenotype as an AcD cell. In contrast, neurons with perisomatic terminations display most often an axon originating from the soma. Our data contribute to the emerging understanding that AIS morphology is highly variable, and potentially a function of the cell type.

Ethanol and age-induced pathologies of the Purkinje neuron (PN) may result from histone deacetylases (HDACs), enzymes which repress transcription through coiling of the DNA. The purposes of this study were to investigate expression patterns of Class 1 and IIa HDACs in PN and the effects of aging and alcohol on the density of HDACs and histone acetylation in PN. Ninety, eight month old rats (30/diet) were fed a liquid ethanol, liquid control, or rat chow diet for 10, 20, or 40weeks (30/treatment duration). Double immunocytochemical labeling on tissue sections from these rats used antibodies against HDAC isoforms or acetylated histones, and calbindin, a marker for PN. Fluorescent intensities were also measured. Results showed a significant age but not an alcohol-related decrease in the densities of HDACs 2, 3, and 7. In contrast, there were age related-increases in the densities of phosphorylated form of HDAC (4, 5, 7) PN and in PN nuclei expressing HDAC 7. There were also a trend towards ethanol-induced inhibition of acetylation as the density of AH2b PN nuclei and AH3 and AH2b fluorescent intensity was significantly lower in the EF compared to the PF rats. This study presents unique data concerning which HDACs are commonly expressed in PN and indicates that aging rather than lengthy alcohol expression alters expression of the HDACs studied here. These results also suggest that lengthy ethanol consumption may inhibit histone deacetylation in PN.

Regulation of the neuron-glia cell-fate switch is a critical step in the development of the CNS. Previously, we demonstrated that Lhx2 is a necessary and sufficient regulator of this process in the mouse hippocampal primordium, such that Lhx2 overexpression promotes neurogenesis and suppresses gliogenesis, whereas loss of Lhx2 has the opposite effect. We tested a series of transcription factors for their ability to mimic Lhx2 overexpression and suppress baseline gliogenesis, and also to compensate for loss of Lhx2 and suppress the resulting enhanced level of gliogenesis in the hippocampus. Here, we demonstrate a novel function of Dmrt5/Dmrta2 as a neurogenic factor in the developing hippocampus. We show that Dmrt5, as well as known neurogenic factors Neurog2 and Pax6, can each not only mimic Lhx2 overexpression, but also can compensate for loss of Lhx2 to different extents. We further uncover a reciprocal regulatory relationship between Dmrt5 and Lhx2, such that each can compensate for loss of the other. Dmrt5 and Lhx2 also have opposing regulatory control on Pax6 and Neurog2, indicating a complex bidirectionally regulated network that controls the neuron-glia cell-fate switch.SIGNIFICANCE STATEMENT We identify Dmrt5 as a novel regulator of the neuron-glia cell-fate switch in the developing hippocampus. We demonstrate Dmrt5 to be neurogenic, and reciprocally regulated by Lhx2: loss of either factor promotes gliogenesis; overexpression of either factor suppresses gliogenesis and promotes neurogenesis; each can substitute for loss of the other. Furthermore, each factor has opposing effects on established neurogenic genes Neurog2 and Pax6 Dmrt5 is known to suppress their expression, and we show that Lhx2 is required to maintain it. Our study reveals a complex regulatory network with bidirectional control of a fundamental feature of CNS development, the control of the production of neurons versus astroglia in the developing hippocampus.Finally, we confirm that Lhx2 binds a highly conserved putative enhancer of Dmrt5, suggesting an evolutionarily conserved regulatory relationship between these factors. Our findings uncover a complex network that involves Lhx2, Dmrt5, Neurog2, and Pax6, and that ensures the appropriate amount and timing of neurogenesis and gliogenesis in the developing hippocampus.

Cholesterol is a critical nutrient requiring tight constraint in the endoplasmic reticulum (ER) due to its uniquely challenging biophysical properties. While the mechanisms by which the ER defends against cholesterol insufficiency are well described, it remains unclear how the ER senses and effectively defends against cholesterol excess. Here, we identify the ER-bound transcription factor nuclear factor erythroid 2 related factor-1, Nrf1/Nfe2L1, as a critical mediator of this process. We show that Nrf1 directly binds to and specifically senses cholesterol in the ER through a defined domain and that cholesterol regulates Nrf1 turnover, processing, localization, and activity. In Nrf1 deficiency, in vivo cholesterol challenges induce massive hepatic cholesterol accumulation and damage, which is rescued by replacing Nrf1 exogenously. This Nrf1-mediated mechanism involves the suppression of CD36-driven inflammatory signaling and derepression of liver X receptor activity. These findings reveal Nrf1 as a guardian of cholesterol homeostasis and a core component of adaptive responses to excess cellular cholesterol.

Melanoma is one of the deadliest cancers, yet the cells of origin and mechanisms of tumor initiation remain unclear. The majority of melanomas emerge from clear skin without a precursor lesion, but it is unknown whether these melanomas can arise from melanocyte stem cells (MCSCs). Here we employ mouse models to define the role of MCSCs as melanoma cells of origin, demonstrate that MCSC quiescence acts as a tumor suppressor, and identify the extrinsic environmental and molecular factors required for the critical early steps of melanoma initiation. Specifically, melanomas originate from melanoma-competent MCSCs upon stimulation by UVB, which induces MCSC activation and translocation via an inflammation-dependent process. Moreover, the chromatin-remodeling factor Hmga2 in the skin plays a critical role in UVB-mediated melanomagenesis. These findings delineate melanoma formation from melanoma-competent MCSCs following extrinsic stimuli, and they suggest that abrogation of Hmga2 function in the microenvironment can suppress MCSC-originating cutaneous melanomas.

Several techniques have been developed to manipulate gene expression temporally in intact neural circuits. However, the applicability of current tools developed for in vivo studies in Drosophila is limited by their incompatibility with existing GAL4 lines and side effects on physiology and behavior. To circumvent these limitations, we adopted a strategy to reversibly regulate protein degradation with a small molecule by using a destabilizing domain (DD). We show that this system is effective across different tissues and developmental stages. We further show that this system can be used to control in vivo gene expression levels with low background, large dynamic range, and in a reversible manner without detectable side effects on the lifespan or behavior of the animal. Additionally, we engineered tools for chemically controlling gene expression (GAL80-DD) and recombination (FLP-DD). We demonstrate the applicability of this technology in manipulating neuronal activity and for high-efficiency sparse labeling of neuronal populations.

In animal cells, nuclear envelope breakdown (NEBD) is required for proper chromosome segregation. Whereas mitotic kinases have been implicated in NEBD, how they coordinate their activity to trigger this event is unclear. Here, we show that both in human cells and Caenorhabditis elegans, the Polo-like kinase 1 (PLK-1) is recruited to the nuclear pore complexes, just prior to NEBD, through its Polo-box domain (PBD). We provide evidence that PLK-1 localization to the nuclear envelope (NE) is required for efficient NEBD. We identify the central channel nucleoporins NPP-1/Nup58, NPP-4/Nup54, and NPP-11/Nup62 as the critical factors anchoring PLK-1 to the NE in C. elegans. In particular, NPP-1, NPP-4, and NPP-11 primed at multiple Polo-docking sites by Cdk1 and PLK-1 itself physically interact with the PLK-1 PBD. We conclude that nucleoporins play an unanticipated regulatory role in NEBD, by recruiting PLK-1 to the NE thereby facilitating phosphorylation of critical downstream targets.

Mobilized peripheral blood (MPB) CD34+ cells were differentiated to CD34+/CD41+ megakaryoblasts. Cells were sorted to obtain a pure megakaryoblast population which was reprogrammed with a hOKSM self-silencing polycistronic lentiviral vector. Resulting iPSC showed normal karyotype and expression of pluripotency associated markers and in vitro spontaneous differentiation towards the 3 germ layers confirmed pluripotency of iPSC lines. Besides normal iPSC applications, these lines can be used as a control line for other megakaryoid origin iPSC and could be applied for epigenetic based research.

The functional variety in neuronal composition of an adult brain is established during development. Recent studies proposed that interactions between genetic intrinsic programs and external cues are necessary to generate proper neural diversity [1]. However, the molecular mechanisms underlying this developmental process are still poorly understood. Three main subtypes of Drosophila mushroom body (MB) neurons are sequentially generated during development and provide a good example of developmental neural plasticity [2]. Our present data propose that the environmentally controlled steroid hormone ecdysone functions as a regulator of early-born MB neuron fate during larval-pupal transition. We found that the BTB-zinc finger factor Chinmo acts upstream of ecdysone signaling to promote a neuronal fate switch. Indeed, Chinmo regulates the expression of the ecdysone receptor B1 isoform to mediate the production of γ and α'β' MB neurons. In addition, we provide genetic evidence for a regulatory negative feedback loop driving the α'β' to αβ MB neuron transition in which ecdysone signaling in turn controls microRNA let-7 depression of Chinmo expression. Thus, our results uncover a novel interaction in the MB neural specification pathway for temporal control of neuronal identity by interplay between an extrinsic hormonal signal and an intrinsic transcription factor cascade.

Peripheral blood mononuclear cells were isolated and cultured to a pure pro-EBL population and reprogrammed using episomal plasmids. The pluripotency of transgene-free induced pluripotent stem cell (iPSC) line was verified by the expression of pluripotency-associated markers and by in vitro spontaneous differentiation towards the 3 germ layers. The iPSC line showed normal karyotype. Peripheral blood is a non-invasive easy accessible cell source and combined with EBL outgrowth in vitro, a routine process obtaining sufficient amount of homogenous cells can be obtained within a week. Using episomal delivery, pro-EBLs can be reprogrammed in a transgene-free, cost effective system.

RING and U-box E3 ubiquitin ligases regulate diverse eukaryotic processes and have been implicated in numerous diseases, but targeting these enzymes remains a major challenge. We report the development of three ubiquitin variants (UbVs), each binding selectively to the RING or U-box domain of a distinct E3 ligase: monomeric UBE4B, phosphorylated active CBL, or dimeric XIAP. Structural and biochemical analyses revealed that UbVs specifically inhibited the activity of UBE4B or phosphorylated CBL by blocking the E2∼Ub binding site. Surprisingly, the UbV selective for dimeric XIAP formed a dimer to stimulate E3 activity by stabilizing the closed E2∼Ub conformation. We further verified the inhibitory and stimulatory functions of UbVs in cells. Our work provides a general strategy to inhibit or activate RING/U-box E3 ligases and provides a resource for the research community to modulate these enzymes.

To ensure the completion of DNA replication and maintenance of genome integrity, DNA repair factors protect stalled replication forks upon replication stress. Previous studies have identified a critical role for the tumor suppressors BRCA1 and BRCA2 in preventing the degradation of nascent DNA by the MRE11 nuclease after replication stress. Here we show that depletion of SMARCAL1, a SNF2-family DNA translocase that remodels stalled forks, restores replication fork stability and reduces the formation of replication stress-induced DNA breaks and chromosomal aberrations in BRCA1/2-deficient cells. In addition to SMARCAL1, other SNF2-family fork remodelers, including ZRANB3 and HLTF, cause nascent DNA degradation and genomic instability in BRCA1/2-deficient cells upon replication stress. Our observations indicate that nascent DNA degradation in BRCA1/2-deficient cells occurs as a consequence of MRE11-dependent nucleolytic processing of reversed forks generated by fork remodelers. These studies provide mechanistic insights into the processes that cause genome instability in BRCA1/2-deficient cells.

Peripheral blood mononuclear cells were isolated from an individual harboring a heterozygous c.859C→T p.Q287* mutation in GFI1B, causing an autosomal dominant bleeding disorder, platelet type, 17 (BDPLT17). PBMCs were differentiated to erythroblasts and reprogrammed by lentiviral delivery of a self-silencing hOKSM polycistronic vector. Pluripotency of iPSC line was confirmed by expression of associated markers and by in vitro spontaneous differentiation towards the 3 germ layers. Normal karyotype confirmed the genomic integrity of iPSCs and the presence of disease causing mutation was shown by Sanger sequencing. The generated iPSCs can be used to study BDPLT17 pathophysiology and basic functions of GFI1B.

Taste stimuli are transduced by taste buds and transmitted to the brain via afferent gustatory fibers. Renewal of taste receptor cells from actively dividing progenitors is finely tuned to maintain taste sensitivity throughout life. We show that conditional β-catenin deletion in mouse taste progenitors leads to rapid depletion of progenitors and Shh+ precursors, which in turn causes taste bud loss, followed by loss of gustatory nerve fibers. In addition, our data suggest LEF1, TCF7 and Wnt3 are involved in a Wnt pathway regulatory feedback loop that controls taste cell renewal in the circumvallate papilla epithelium. Unexpectedly, taste bud decline is greater in the anterior tongue and palate than in the posterior tongue. Mutant mice with this regional pattern of taste bud loss were unable to discern sweet at any concentration, but could distinguish bitter stimuli, albeit with reduced sensitivity. Our findings are consistent with published reports wherein anterior taste buds have higher sweet sensitivity while posterior taste buds are better tuned to bitter, and suggest β-catenin plays a greater role in renewal of anterior versus posterior taste buds.

Glucose constitutes a major source of energy of mammalian brains. Glucose uptake at the blood-brain barrier (BBB) occurs through a facilitated glucose transport, through glucose transporter 1 (GLUT1), although other isoforms have been described at the BBB. Mutations in GLUT1 are associated with the GLUT1 deficiency syndrome, yet none of the current in vitro models of the human BBB maybe suited for modeling such a disorder. In this study, we investigated the expression of glucose transporters and glucose diffusion across brain microvascular endothelial cells (BMECs) derived from healthy patient-derived induced pluripotent stem cells (iPSCs). We investigated the expression of different glucose transporters at the BBB using immunocytochemistry and flow cytometry and measured glucose uptake and diffusion across BMEC monolayers obtained from two iPSC lines and from hCMEC/D3 cells. BMEC monolayers showed expression of several glucose transporters, in particular GLUT1, GLUT3, and GLUT4. Diffusion of glucose across the monolayers was mediated via a saturable transcellular mechanism and partially inhibited by pharmacological inhibitors. Taken together, our study suggests the presence of several glucose transporters isoforms at the human BBB and demonstrates the feasibility of modeling glucose across the BBB using patient-derived stem cells.

Vascular endothelial growth factor (VEGF) is a major driver of blood vessel formation. However, the signal transduction pathways culminating in the biological consequences of VEGF signaling are only partially understood. Here, we show that the Hippo pathway effectors YAP and TAZ work as crucial signal transducers to mediate VEGF-VEGFR2 signaling during angiogenesis. We demonstrate that YAP/TAZ are essential for vascular development as endothelium-specific deletion of YAP/TAZ leads to impaired vascularization and embryonic lethality. Mechanistically, we show that VEGF activates YAP/TAZ via its effects on actin cytoskeleton and that activated YAP/TAZ induce a transcriptional program to further control cytoskeleton dynamics and thus establish a feedforward loop that ensures a proper angiogenic response. Lack of YAP/TAZ also results in altered cellular distribution of VEGFR2 due to trafficking defects from the Golgi apparatus to the plasma membrane. Altogether, our study identifies YAP/TAZ as central mediators of VEGF signaling and therefore as important regulators of angiogenesis.

Duplication 15q syndrome (Dup15q) is an autism-associated disorder co-incident with high rates of pediatric epilepsy. Additional copies of the E3 ubiquitin ligase UBE3A are thought to cause Dup15q phenotypes, yet models overexpressing UBE3A in neurons have not recapitulated the epilepsy phenotype. We show that Drosophila endogenously expresses Dube3a (fly UBE3A homolog) in glial cells and neurons, prompting an investigation into the consequences of glial Dube3a overexpression. Here we expand on previous work showing that the Na+/K+ pump ATPα is a direct ubiquitin ligase substrate of Dube3a. A robust seizure-like phenotype was observed in flies overexpressing Dube3a in glial cells, but not neurons. Glial-specific knockdown of ATPα also produced seizure-like behavior, and this phenotype was rescued by simultaneously overexpressing ATPα and Dube3a in glia. Our data provides the basis of a paradigm shift in Dup15q research given that clinical phenotypes have long been assumed to be due to neuronal UBE3A overexpression.

Using Nogo antibodies with defined binding specificity, Nogo-B, but not Nogo-A, was localized on radial glia in the floor plate of mouse embryos. The presence of Nogo-B was confirmed in Nogo-A knockout mice. In explant cultures of embryonic day (E) 11 and E12 spinal cord, blocking of NgR function with antagonist peptide NEP1-40 reduced the crossing of newly arrived commissural axons, resulting in an accumulation of growth cones in the floor plate. Analysis of growth cone morphology demonstrated an increase in size of growth cones in the floor plate after peptide treatment, which was not detected in axons growing toward the midline. In knockout embryos, midline crossing was not affected by absence of Nogo-A. In co-culture experiments using collagen gel, floor plate showed a strong inhibitory effect on the extension of post-commissural neurites from the spinal cord. This effect was abolished by NEP1-40, and was observed neither in pre-commissural neurites, nor in post-commissural neurites grown with floor plate derived from Nogo-A knockout embryo. Furthermore, western blot analysis of conditioned medium from floor plates showed a truncated form of Nogo with molecular weight of 37 kDa, which could mediate the diffusible effect to axon growth. We conclude that Nogo-B is expressed in the floor plate of mouse embryo, which probably mediates axon crossing in the spinal cord by repelling axons out of the midline when they start upregulate NgR. Nogo acts on axon growth not only through a contact-mediated mechanism, but also through a diffusible mechanism.

Formation and expression of memories are critical for context-dependent decision making. In Drosophila, a courting male rejected by a mated female subsequently courts less avidly when paired with a virgin female, a behavioral modification attributed to "courtship memory." Here we show the critical role of hormonal state for maintenance of courtship memory. Ecdysis-triggering hormone (ETH) is essential for courtship memory through regulation of juvenile hormone (JH) levels in adult males. Reduction of JH levels via silencing of ETH signaling genes impairs short-term courtship memory, a phenotype rescuable by the JH analog methoprene. JH-deficit-induced memory impairment involves rapid decay rather than failure of memory acquisition. A critical period governs memory performance during the first 3 days of adulthood. Using sex-peptide-expressing "pseudo-mated" trainers, we find that robust courtship memory elicited in the absence of aversive chemical mating cues also is dependent on ETH-JH signaling. Finally, we find that JH acts through dopaminergic neurons and conclude that an ETH-JH-dopamine signaling cascade is required during a critical period for promotion of social-context-dependent memory.

Really interesting new gene (RING) finger protein is a type of zinc-binding motif found in a large family of functionally distinct proteins. RING finger proteins are involved in diverse cellular processes including apoptosis, DNA repair, cell cycle, signal transduction, tumour suppressor, vesicular transport, and peroxisomal biogenesis. RING finger protein 38 (RNF38) is a member of the family whose functions remain unknown. To gain insight into the putative effects of RNF38 in the central nervous system, we localised its expression. The aim of this study was to identify the neuroanatomical location(s) of rnf38 mRNA and its peptide, determine the type of RNF38-expressing cells, and measure rnf38 gene expression in the brain of male tilapia. The distributions of rnf38 mRNA and its peptide were visualised using in situ hybridisation with digoxigenin-labelled RNA antisense and immunocytochemistry, respectively. Both were identically distributed throughout the brain, including the telencephalon, preoptic area, optic tectum, hypothalamus, cerebellum, and the hindbrain. Double-labelling immunocytochemistry for RNF38 and the neuronal marker HuC/D showed that most but not all RNF38 protein was expressed in neuronal nuclei. Quantitative real-time polymerase chain reaction showed the highest level of rnf38 mRNA in the midbrain, followed by the preoptic area, cerebellum, optic tectum, telencephalon, hindbrain and hypothalamus. These findings reveal a differential spatial pattern of RNF38 in the tilapia brain, suggesting that it has potentially diverse functions related to neuronal activity.

The recycling of neurotransmitters is essential for sustained synaptic transmission. In Drosophila, histamine recycling is required for visual synaptic transmission. Synaptic histamine is rapidly taken up by laminar glia, and is converted to carcinine. After delivered back to photoreceptors, carcinine is hydrolyzed to release histamine and β-alanine. This histamine is repackaged into synaptic vesicles, but it is unclear how the β-alanine is returned to the laminar glial cells. Here, we identified a new β-alanine transporter, which we named BalaT (Beta-alanine Transporter). Null balat mutants exhibited lower levels of β-alanine, as well as less β-alanine accumulation in the retina. Moreover, BalaT is expressed and required in retinal pigment cells for maintaining visual synaptic transmission and phototaxis behavior. These results provide the first genetic evidence that retinal pigment cells play a critical role in visual neurotransmission, and suggest that a BalaT-dependent β-alanine trafficking pathway is required for histamine homeostasis and visual neurotransmission.

Input-timing-dependent plasticity (ITDP) is a circuit-based synaptic learning rule by which paired activation of entorhinal cortical (EC) and Schaffer collateral (SC) inputs to hippocampal CA1 pyramidal neurons (PNs) produces a long-term enhancement of SC excitation. We now find that paired stimulation of EC and SC inputs also induces ITDP of SC excitation of CA2 PNs. However, whereas CA1 ITDP results from long-term depression of feedforward inhibition (iLTD) as a result of activation of CB1 endocannabinoid receptors on cholecystokinin-expressing interneurons, CA2 ITDP results from iLTD through activation of δ-opioid receptors on parvalbumin-expressing interneurons. Furthermore, whereas CA1 ITDP has been previously linked to enhanced specificity of contextual memory, we find that CA2 ITDP is associated with enhanced social memory. Thus, ITDP may provide a general synaptic learning rule for distinct forms of hippocampal-dependent memory mediated by distinct hippocampal regions.

Reck, a GPI-anchored membrane protein, and Gpr124, an orphan GPCR, have been implicated in Wnt7a/Wnt7b signaling in the CNS vasculature. We show here that vascular endothelial cell (EC)-specific reduction in Reck impairs CNS angiogenesis and that EC-specific postnatal loss of Reck, combined with loss of Norrin, impairs blood-brain barrier (BBB) maintenance. The most N-terminal domain of Reck binds to the leucine-rich repeat (LRR) and immunoglobulin (Ig) domains of Gpr124, and weakening this interaction by targeted mutagenesis reduces Reck/Gpr124 stimulation of Wnt7a signaling in cell culture and impairs CNS angiogenesis. Finally, a soluble Gpr124(LRR-Ig) probe binds to cells expressing Frizzled, Wnt7a or Wnt7b, and Reck, and a soluble Reck(CC1-5) probe binds to cells expressing Frizzled, Wnt7a or Wnt7b, and Gpr124. These experiments indicate that Reck and Gpr124 are part of the cell surface protein complex that transduces Wnt7a- and Wnt7b-specific signals in mammalian CNS ECs to promote angiogenesis and regulate the BBB.

Angelman syndrome (AS) is a debilitating neurodevelopmental disorder caused by loss of function of the maternally inherited UBE3A allele. It is currently unclear how the consequences of this genetic insult unfold to impair neurodevelopment. We reasoned that by elucidating the basis of microcephaly in AS, a highly penetrant syndromic feature with early postnatal onset, we would gain new insights into the mechanisms by which maternal UBE3A loss derails neurotypical brain growth and function. Detailed anatomical analysis of both male and female maternal Ube3a-null mice reveals that microcephaly in the AS mouse model is primarily driven by deficits in the growth of white matter tracts, which by adulthood are characterized by densely packed axons of disproportionately small caliber. Our results implicate impaired axon growth in the pathogenesis of AS and identify noninvasive structural neuroimaging as a potentially valuable tool for gauging therapeutic efficacy in the disorder.SIGNIFICANCE STATEMENT People who maternally inherit a deletion or nonfunctional copy of the UBE3A gene develop Angelman syndrome (AS), a severe neurodevelopmental disorder. To better understand how loss of maternal UBE3A function derails brain development, we analyzed brain structure in a maternal Ube3a knock-out mouse model of AS. We report that the volume of white matter (WM) is disproportionately reduced in AS mice, indicating that deficits in WM development are a major factor underlying impaired brain growth and microcephaly in the disorder. Notably, we find that axons within the WM pathways of AS model mice are abnormally small in caliber. This defect is associated with slowed nerve conduction, which could contribute to behavioral deficits in AS, including motor dysfunction.

Conflicts between transcription and replication are a potent source of DNA damage. Co-transcriptional R-loops could aggravate such conflicts by creating an additional barrier to replication fork progression. Here, we use a defined episomal system to investigate how conflict orientation and R-loop formation influence genome stability in human cells. R-loops, but not normal transcription complexes, induce DNA breaks and orientation-specific DNA damage responses during conflicts with replication forks. Unexpectedly, the replisome acts as an orientation-dependent regulator of R-loop levels, reducing R-loops in the co-directional (CD) orientation but promoting their formation in the head-on (HO) orientation. Replication stress and deregulated origin firing increase the number of HO collisions leading to genome-destabilizing R-loops. Our findings connect DNA replication to R-loop homeostasis and suggest a mechanistic basis for genome instability resulting from deregulated DNA replication, observed in cancer and other disease states.

Homocysteine, a metabolite of the methionine cycle has been reported to play a role in neurotoxicity through activation of N-methyl-d-aspartate receptors (NMDAR)-mediated signaling pathway. The proposed mechanisms associated with homocysteine-NMDAR-induced neurotoxicity involve a unique signaling pathway that triggers a crosstalk between extracellular signal-regulated kinase (ERK) and p38 MAPKs, where activation of p38 MAPK is downstream of and dependent on ERK MAPK. However, the molecular basis of the ERK MAPK-mediated p38 MAPK activation is not understood. This study investigates whether α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) play a role in facilitating the ERK MAPK-mediated p38 MAPK activation. Using surface biotinylation and immunoblotting approaches we show that treatment with homocysteine leads to a decrease in surface expression of GluA2-AMPAR subunit in neurons, but have no effect on the surface expression of GluA1-AMPAR subunit. Inhibition of NMDAR activation with D-AP5 or ERK MAPK phosphorylation with PD98059 attenuates homocysteine-induced decrease in surface expression of GluA2-AMPAR subunit. The decrease in surface expression of GluA2-AMPAR subunit is associated with p38 MAPK phosphorylation, which is inhibited by 1-napthyl acetyl spermine trihydrochloride (NASPM), a selective antagonist of GluA2-lacking Ca2+ -permeable AMPARs. These results suggest that homocysteine-NMDAR-mediated ERK MAPK phosphorylation leads to a decrease in surface expression of GluA2-AMPAR subunit resulting in Ca2+ influx through the GluA2-lacking Ca2+ -permeable AMPARs and p38 MAPK phosphorylation. Cell death assays further show that inhibition of AMPAR activity with 2,3-dioxo-6-nitro-1,2,3,4,tetrahydrobenzoquinoxaline-7-sulfonamide (NBQX)/6-cyano-7-nitroquinoxaline-2,3, -dione (CNQX) or GluA2-lacking Ca2+ -permeable AMPAR activity with NASPM attenuates homocysteine-induced neurotoxicity. We have identified an important mechanism involved in homocysteine-induced neurotoxicity that highlights the intermediary role of GluA2-lacking Ca2+ -permeable AMPARs in the crosstalk between ERK and p38 MAPKs.

Hypothalamic agouti-related peptide (AgRP) neurons potently stimulate food intake, whereas proopiomelanocortin (POMC) neurons inhibit feeding. Whether AgRP neurons exert their orexigenic actions, at least in part, by inhibiting anorexigenic POMC neurons remains unclear. Here, the connectivity between GABA-releasing AgRP neurons and POMC neurons was examined in brain slices from male and female mice. GABA-mediated spontaneous IPSCs (sIPSCs) in POMC neurons were unaffected by disturbing GABA release from AgRP neurons either by cell type-specific deletion of the vesicular GABA transporter or by expression of botulinum toxin in AgRP neurons to prevent vesicle-associated membrane protein 2-dependent vesicle fusion. Additionally, there was no difference in the ability of μ-opioid receptor (MOR) agonists to inhibit sIPSCs in POMC neurons when MORs were deleted from AgRP neurons, and activation of the inhibitory designer receptor hM4Di on AgRP neurons did not affect sIPSCs recorded from POMC neurons. These approaches collectively indicate that AgRP neurons do not significantly contribute to the strong spontaneous GABA input to POMC neurons. Despite these observations, optogenetic stimulation of AgRP neurons reliably produced evoked IPSCs in POMC neurons, leading to the inhibition of POMC neuron firing. Thus, AgRP neurons can potently affect POMC neuron function without contributing a significant source of spontaneous GABA input to POMC neurons. Together, these results indicate that the relevance of GABAergic inputs from AgRP to POMC neurons is state dependent and highlight the need to consider different types of transmitter release in circuit mapping and physiologic regulation.SIGNIFICANCE STATEMENT Agouti-related peptide (AgRP) neurons play an important role in driving food intake, while proopiomelanocortin (POMC) neurons inhibit feeding. Despite the importance of these two well characterized neuron types in maintaining metabolic homeostasis, communication between these cells remains poorly understood. To provide clarity to this circuit, we made electrophysiological recordings from mouse brain slices and found that AgRP neurons do not contribute spontaneously released GABA onto POMC neurons, although when activated with channelrhodopsin AgRP neurons inhibit POMC neurons through GABA-mediated transmission. These findings indicate that the relevance of AgRP to POMC neuron GABA connectivity depends on the state of AgRP neuron activity and suggest that different types of transmitter release should be considered when circuit mapping.

Simultaneous hyperactivation of Wnt and antioxidant response (AR) are often observed during oncogenesis. However, it remains unclear how the β-catenin-driven Wnt and the Nrf2-driven AR mutually regulate each other. The situation is compounded because many players in these two pathways are redox sensors, rendering bolus redox signal-dosing methods uninformative. Herein we examine the ramifications of single-protein target-specific AR upregulation in various knockdown lines. Our data document that Nrf2/AR strongly inhibits β-catenin/Wnt. The magnitude and mechanism of this negative regulation are dependent on the direct interaction between β-catenin N terminus and β-TrCP1 (an antagonist of both Nrf2 and β-catenin), and independent of binding between Nrf2 and β-TrCP1. Intriguingly, β-catenin positively regulates AR. Because AR is a negative regulator of Wnt regardless of β-catenin N terminus, this switch of function is likely sufficient to establish a new Wnt/AR equilibrium during tumorigenesis.

Ischemic stroke is a leading cause of adult disability with no pharmacological treatments to promote the recovery of lost function. Neutralizing antibodies against the neurite outgrowth inhibitor Nogo-A have emerged as a promising treatment for subacute and chronic stroke in animal models; however, whether anti-Nogo-A treatment affects poststroke neurogenesis remains poorly understood. In this study, we confirmed expression of Nogo-A by neuroblasts in the adult rat subventricular zone (SVZ), a major neurogenic niche; however, we found no evidence that Nogo-A was expressed at the surface of these cells. In vitro migration assays demonstrated that Nogo-A signaling induced a modest reduction in neuroblast migration speed, while anti-Nogo-A antibodies had no effect on motility properties. Using a permanent distal middle cerebral artery occlusion model of cortical stroke, we found that the number of proliferating cells in the SVZ was unaffected in response to stroke, while neuroblast mobilization from the SVZ toward the stroke lesion correlated positively with lesion size. However, we found no evidence that proliferation or neuroblast mobilization were affected by anti-Nogo-A antibody treatment. Our results suggest that the SVZ is not a therapeutic target of anti-Nogo-A immunotherapy, and contribute to our understanding of the SVZ response to cortical stroke.

E-Cadherin is a major component of adherens junctions on cell surfaces. SNX16 is a unique member of sorting nexins that contains a coiled-coil (CC) domain downstream of the PX domain. We report here that SNX16 regulates the recycling trafficking of E-cadherin. We solved the crystal structure of PX-CC unit of SNX16 and revealed a unique shear shaped homodimer. We identified a novel PI3P binding pocket in SNX16 that consists of both the PX and the CC domains. Surprisingly, we showed that the PPII/α2 loop, which is generally regarded as a membrane insertion loop in PX family proteins, is involved in the E-cadherin binding with SNX16. We then proposed a multivalent membrane binding model for SNX16. Our study postulates a new mechanism for coordinated membrane binding and cargo binding for SNX family proteins in general, and provide novel insights into recycling trafficking of E-cadherin.

Diffuse neuromodulatory systems such as norepinephrine (NE) control brain-wide states such as arousal, but whether they control complex social behaviors more specifically is not clear. Octopamine (OA), the insect homolog of NE, is known to promote both arousal and aggression. We have performed a systematic, unbiased screen to identify OA receptor-expressing neurons (OARNs) that control aggression in Drosophila. Our results uncover a tiny population of male-specific aSP2 neurons that mediate a specific influence of OA on aggression, independent of any effect on arousal. Unexpectedly, these neurons receive convergent input from OA neurons and P1 neurons, a population of FruM+ neurons that promotes male courtship behavior. Behavioral epistasis experiments suggest that aSP2 neurons may constitute an integration node at which OAergic neuromodulation can bias the output of P1 neurons to favor aggression over inter-male courtship. These results have potential implications for thinking about the role of related neuromodulatory systems in mammals.

Extracellular signals are transduced to the cell nucleus by effectors that bind to enhancer complexes to operate transcriptional switches. For example, the Wnt enhanceosome is a multiprotein complex associated with Wnt-responsive enhancers through T cell factors (TCF) and kept silent by Groucho/TLE co-repressors. Wnt-activated β-catenin binds to TCF to overcome this repression, but how it achieves this is unknown. Here, we discover that this process depends on the HECT E3 ubiquitin ligase Hyd/UBR5, which is required for Wnt signal responses in Drosophila and human cell lines downstream of activated Armadillo/β-catenin. We identify Groucho/TLE as a functionally relevant substrate, whose ubiquitylation by UBR5 is induced by Wnt signaling and conferred by β-catenin. Inactivation of TLE by UBR5-dependent ubiquitylation also involves VCP/p97, an AAA ATPase regulating the folding of various cellular substrates including ubiquitylated chromatin proteins. Thus, Groucho/TLE ubiquitylation by Hyd/UBR5 is a key prerequisite that enables Armadillo/β-catenin to activate transcription.

Active forces generated at kinetochores move chromosomes, and the dynamic spindle must robustly anchor kinetochore fibers (k-fibers) to bear this load. The mammalian spindle bears the load of chromosome movement far from poles, but we do not know where and how-physically and molecularly-this load distributes across the spindle. In part, this is because probing spindle mechanics in live cells is difficult. Yet answering this question is key to understanding how the spindle generates and responds to force and performs its diverse mechanical functions. Here, we map load-bearing across the mammalian spindle in space-time and dissect local anchorage mechanics and mechanism. To do so, we laser-ablate single k-fibers at different spindle locations and in different molecular backgrounds and quantify the immediate relaxation of chromosomes, k-fibers, and microtubule speckles. We find that load redistribution is locally confined in all directions: along the first 3-4 μm from kinetochores, scaling with k-fiber length, and laterally within ∼2 μm of k-fiber sides, without detectable load sharing between neighboring k-fibers. A phenomenological model suggests that dense, transient crosslinks to the spindle along k-fibers bear the load of chromosome movement but that these connections do not limit the timescale of spindle reorganization. The microtubule crosslinker NuMA is needed for the local load-bearing observed, whereas Eg5 and PRC1 are not detectably required, suggesting specialization in mechanical function. Together, the data and model suggest that NuMA-mediated crosslinks locally bear load, providing mechanical isolation and redundancy while allowing spindle fluidity. These features are well suited to support robust chromosome segregation.

In most sexually reproducing plants, a single somatic, sub-epidermal cell in an ovule is selected to differentiate into a megaspore mother cell, which is committed to giving rise to the female germline. However, it remains unclear how intercellular signaling among somatic cells results in only one cell in the sub-epidermal layer differentiating into the megaspore mother cell. Here we uncovered a role of the THO complex in restricting the megaspore mother cell fate to a single cell. Mutations in TEX1, HPR1, and THO6, components of the THO/TREX complex, led to the formation of multiple megaspore mother cells, which were able to initiate gametogenesis. We demonstrated that TEX1 repressed the megaspore mother cell fate by promoting the biogenesis of TAS3-derived trans-acting small interfering RNA (ta-siRNA), which represses ARF3 expression. The TEX1 protein was present in epidermal cells, but not in the germline, and, through TAS3-derived ta-siRNA, restricted ARF3 expression to the medio domain of ovule primordia. Expansion of ARF3 expression into lateral epidermal cells in a TAS3 ta-siRNA-insensitive mutant led to the formation of supernumerary megaspore mother cells, suggesting that TEX1- and TAS3-mediated restriction of ARF3 expression limits excessive megaspore mother cell formation non-cell-autonomously. Our findings reveal the role of a small-RNA pathway in the regulation of female germline specification in Arabidopsis.

A large proportion of critically ill patients have alterations in the hypothalamus-pituitary-thyroid (HPT) axis, collectively known as the nonthyroidal illness syndrome. Nonthyroidal illness syndrome is characterized by low serum thyroid hormone (TH) concentrations accompanied by a suppressed central component of the HPT axis and persistent low serum TSH. In hypothalamic tanycytes, the expression of type 2 deiodinase (D2) is increased in several animal models of inflammation. Because D2 is a major source of T3 in the brain, this response is thought to suppress TRH expression in the paraventricular nucleus via increased local bioavailability of T3. The inflammatory pathway component RelA (the p65 subunit of nuclear factor-κB) can bind the Dio2 promoter and increases D2 expression after lipopolysaccharide (LPS) stimulation in vitro. We aimed to determine whether RelA signaling in tanycytes is essential for the LPS-induced D2 increase in vivo by conditional elimination of RelA in tanycytes of mice (RelA(ASTKO)). Dio2 and Trh mRNA expression were assessed by quantitative in situ hybridization 8 or 24 hours after saline or LPS injection. At the same time points, we measured pituitary Tshβ mRNA expression and serum T3 and T4 concentrations. In RelA(ASTKO) mice the LPS-induced increase in Dio2 and decrease in Trh mRNA levels in the hypothalamus were reduced compared with the wild-type littermates, whereas the drop in pituitary Tshβ expression and in serum TH concentrations persisted. In conclusion, RelA is essential for the LPS-induced hypothalamic D2 increase and TRH decrease. The central changes in the HPT axis are, however, not required for the down-regulation of Tshβ expression and serum TH concentrations.

The trialkyltins tributyltin (TBT) and triphenyltin (TPT) can function as rexinoid-X receptor (RXR) agonists. We recently showed that RXR agonists can alter thyroid hormone (TH) signaling in a mammalian pituitary TH-responsive reporter cell line, GH3.TRE-Luc. The prevalence of TBT and TPT in the environment prompted us to test whether they could also affect TH signaling. Both trialkyltins induced the integrated luciferase reporter alone and potentiated TH activation at low doses. Trimethyltin, which is not an RXR agonist, did not. We turned to a simple, robust, and specific in vivo model system of TH action: metamorphosis of Xenopus laevis, the African clawed frog. Using a precocious metamorphosis assay, we found that 1nM TBT and TPT, but not trimethyltin, greatly potentiated the effect of TH treatment on resorption phenotypes of the tail, which is lost at metamorphosis, and in the head, which undergoes extensive remodeling including gill loss. Consistent with these responses, TH-induced caspase-3 activation in the tail was enhanced by cotreatment with TBT. Induction of a transgenic reporter gene and endogenous collagenase 3 (mmp13) and fibroblast-activating protein-α (fap) genes were not induced by TBT alone, but TH induction was significantly potentiated by TBT. However, induction of other TH receptor target genes such as TRβ and deiodinase 3 by TH were not affected by TBT cotreatment. These data indicate that trialkyltins that can function as RXR agonists can selectively potentiate gene expression and resultant morphological programs directed by TH signaling in vivo.

Clearance of bacteria by macrophages involves internalization of the microorganisms into phagosomes, which are then delivered to endolysosomes for enzymatic degradation. These spatiotemporally segregated processes are not known to be functionally coupled. Here, we show that lysosomal degradation of bacteria sustains phagocytic uptake. In Drosophila and mammalian macrophages, lysosomal dysfunction due to loss of the endolysosomal Cl- transporter ClC-b/CLCN7 delayed degradation of internalized bacteria. Unexpectedly, defective lysosomal degradation of bacteria also attenuated further phagocytosis, resulting in elevated bacterial load. Exogenous application of bacterial peptidoglycans restored phagocytic uptake in the lysosomal degradation-defective mutants via a pathway requiring cytosolic pattern recognition receptors and NF-κB. Mammalian macrophages that are unable to degrade internalized bacteria also exhibit compromised NF-κB activation. Our findings reveal a role for phagolysosomal degradation in activating an evolutionarily conserved signaling cascade, which ensures that continuous uptake of bacteria is preceded by lysosomal degradation of microbes.

Neutrophils release neutrophil extracellular traps (NETs) which ensnare pathogens and have pathogenic functions in diverse diseases. We examined the NETosis pathways induced by five stimuli; PMA, the calcium ionophore A23187, nigericin, Candida albicans and Group B Streptococcus. We studied NET production in neutrophils from healthy donors with inhibitors of molecules crucial to PMA-induced NETs including protein kinase C, calcium, reactive oxygen species, the enzymes myeloperoxidase (MPO) and neutrophil elastase. Additionally, neutrophils from chronic granulomatous disease patients, carrying mutations in the NADPH oxidase complex or a MPO-deficient patient were examined. We show that PMA, C. albicans and GBS use a related pathway for NET induction, whereas ionophores require an alternative pathway but that NETs produced by all stimuli are proteolytically active, kill bacteria and composed mainly of chromosomal DNA. Thus, we demonstrate that NETosis occurs through several signalling mechanisms, suggesting that extrusion of NETs is important in host defence.

Alzheimer's disease (AD) is characterized by progressive cognitive decline, increasingly attributed to neuronal dysfunction induced by amyloid-β oligomers (AβOs). Although the impact of AβOs on neurons has been extensively studied, only recently have the possible effects of AβOs on astrocytes begun to be investigated. Given the key roles of astrocytes in synapse formation, plasticity, and function, we sought to investigate the impact of AβOs on astrocytes, and to determine whether this impact is related to the deleterious actions of AβOs on synapses. We found that AβOs interact with astrocytes, cause astrocyte activation and trigger abnormal generation of reactive oxygen species, which is accompanied by impairment of astrocyte neuroprotective potential in vitro We further show that both murine and human astrocyte conditioned media (CM) increase synapse density, reduce AβOs binding, and prevent AβO-induced synapse loss in cultured hippocampal neurons. Both a neutralizing anti-transforming growth factor-β1 (TGF-β1) antibody and siRNA-mediated knockdown of TGF-β1, previously identified as an important synaptogenic factor secreted by astrocytes, abrogated the protective action of astrocyte CM against AβO-induced synapse loss. Notably, TGF-β1 prevented hippocampal dendritic spine loss and memory impairment in mice that received an intracerebroventricular infusion of AβOs. Results suggest that astrocyte-derived TGF-β1 is part of an endogenous mechanism that protects synapses against AβOs. By demonstrating that AβOs decrease astrocyte ability to protect synapses, our results unravel a new mechanism underlying the synaptotoxic action of AβOs in AD.SIGNIFICANCE STATEMENT Alzheimer's disease is characterized by progressive cognitive decline, mainly attributed to synaptotoxicity of the amyloid-β oligomers (AβOs). Here, we investigated the impact of AβOs in astrocytes, a less known subject. We show that astrocytes prevent synapse loss induced by AβOs, via production of transforming growth factor-β1 (TGF-β1). We found that AβOs trigger morphological and functional alterations in astrocytes, and impair their neuroprotective potential. Notably, TGF-β1 reduced hippocampal dendritic spine loss and memory impairment in mice that received intracerebroventricular infusions of AβOs. Our results describe a new mechanism underlying the toxicity of AβOs and indicate novel therapeutic targets for Alzheimer's disease, mainly focused on TGF-β1 and astrocytes.

The subunit composition of synaptic NMDA receptors (NMDAR), such as the relative content of GluN2A- and GluN2B-containing receptors, greatly influences the glutamate synaptic transmission. Receptor co-agonists, glycine and D-serine, have intriguingly emerged as potential regulators of the receptor trafficking in addition to their requirement for its activation. Using a combination of single-molecule imaging, biochemistry and electrophysiology, we show that glycine and D-serine relative availability at rat hippocampal glutamatergic synapses regulate the trafficking and synaptic content of NMDAR subtypes. Acute manipulations of co-agonist levels, both ex vivo and in vitro, unveil that D-serine alter the membrane dynamics and content of GluN2B-NMDAR, but not GluN2A-NMDAR, at synapses through a process requiring PDZ binding scaffold partners. In addition, using FRET-based FLIM approach, we demonstrate that D-serine rapidly induces a conformational change of the GluN1 subunit intracellular C-terminus domain. Together our data fuels the view that the extracellular microenvironment regulates synaptic NMDAR signaling.

The parasitic wasp, Trichogramma evanescens, is an extremely small insect, with a body length as small as 0.3 mm. To facilitate this miniaturization, their brains may have evolved to contain smaller neural components and/or reduced neural complexity than larger insects. Here, we study whether the size and number of neurons are reduced in the miniaturized brain of T. evanescens, focusing on neurons that express serotonin (5HT), octopamine (OA) and dopamine (DA). We provide the first description of the distribution, projection patterns and number of 5HT-, OA- and DA-like immunoreactive cell bodies in T. evanescens and compare our observations with descriptions of much larger insects. The brains of T. evanescens contain comparable numbers of monoaminergic neurons to those of larger insects. Serotonergic neurons appear to be especially conserved; most of the clusters contain a similar number of neurons to those described in Apis mellifera and Drosophila melanogaster. This maintained complexity may have been facilitated by miniaturization of neuron size. However, many dopaminergic and some octopaminergic neuron clusters in T. evanescens contain fewer neurons than in larger insects. Modification of the complexity of these monoaminergic systems may have been necessary to maintain neuron functionality during brain miniaturization in T. evanescens. Our results reveal some of the evolutionary adaptations that may enable behavioural and cognitive complexity with respect to miniaturized brains.

Chronic exposure to high-saturated fat diets (HFDs) increases the prevalence of obesity and contributes to the development of low-grade inflammation and insulin resistance. A possible mediator accounting for obesity-associated inflammation and insulin resistance is Toll-like receptor 4 (TLR4). We investigated the role of adipocyte TLR4 in lipid and glucose homeostasis through an inducible, adipocyte-specific deletion of TLR4 in a mouse model that is referred to as the "Tadipo" mouse. Consistent with a critical role for inflammation as a positive force for healthy adipose tissue expansion, chronic HFD exposure results in exacerbated whole-body and muscle insulin resistance in the absence of TLR4 in the adipocyte. Elimination of TLR4 in adipocytes affects TLR4 expression in other tissues, with reduced TLR4 expression in peritoneal macrophages and in the liver. In contrast, TLR4 deletion from adipocytes protects whole-body insulin sensitivity after an acute lipid challenge during a hyperinsulinemic euglycemic clamp. Our results therefore demonstrate dichotomous effects of TLR4 on adipose tissue functionality, with an important positive role of TLR4 during a chronic HFD challenge due to the lack of adipose tissue remodeling and a negative role of TLR4 as a mediator of insulin resistance in the adipocyte during an acute challenge with saturated fatty acids.

Touch perception depends on integrating signals from multiple types of peripheral mechanoreceptors. Merkel-cell associated afferents are thought to play a major role in form perception by encoding surface features of touched objects. However, activity of Merkel afferents during active touch has not been directly measured. Here, we show that Merkel and unidentified slowly adapting afferents in the whisker system of behaving mice respond to both self-motion and active touch. Touch responses were dominated by sensitivity to bending moment (torque) at the base of the whisker and its rate of change and largely explained by a simple mechanical model. Self-motion responses encoded whisker position within a whisk cycle (phase), not absolute whisker angle, and arose from stresses reflecting whisker inertia and activity of specific muscles. Thus, Merkel afferents send to the brain multiplexed information about whisker position and surface features, suggesting that proprioception and touch converge at the earliest neural level.

Animals exhibit a behavioral response to novel sensory stimuli about which they have no prior knowledge. We have examined the neural and behavioral correlates of novelty and familiarity in the olfactory system of Drosophila. Novel odors elicit strong activity in output neurons (MBONs) of the α'3 compartment of the mushroom body that is rapidly suppressed upon repeated exposure to the same odor. This transition in neural activity upon familiarization requires odor-evoked activity in the dopaminergic neuron innervating this compartment. Moreover, exposure of a fly to novel odors evokes an alerting response that can also be elicited by optogenetic activation of α'3 MBONs. Silencing these MBONs eliminates the alerting behavior. These data suggest that the α'3 compartment plays a causal role in the behavioral response to novel and familiar stimuli as a consequence of dopamine-mediated plasticity at the Kenyon cell-MBONα'3 synapse.

Sphingosine kinase 2 (SK2) is a ubiquitously expressed lipid kinase that has important, albeit complex and poorly understood, roles in regulating cell survival and cell death. In addition to being able to promote cell cycle arrest and apoptosis under certain conditions, it has recently been shown that SK2 can promote neoplastic transformation and tumorigenesis in vivo. Therefore, well validated and reliable tools are required to study and better understand the true functions of SK2. Here, we compare two commercially available SK2 antibodies: a rabbit polyclonal antibody from Proteintech that recognizes amino acids 266-618 of human SK2a, and a rabbit polyclonal antibody from ECM Biosciences that recognizes amino acids 36-52 of human SK2a. We examine the performance of these antibodies for use in immunoblotting, immunoprecipitation and immunofluorescence staining of endogenous SK2, using human HEK293 and HeLa cell lines, as well as mouse embryonic fibroblasts (MEFs). Furthermore, we assess the specificity of these antibodies to the target protein through the use of siRNA-mediated SK2 knockdown and SK2 knockout ( Sphk2-/-) MEFs. Our results demonstrate that the Proteintech anti-SK2 antibody reproducibly displayed superior sensitivity and selectivity towards SK2 in immunoblot analyses, while the ECM Biosciences anti-SK2 antibody was reproducibly superior for SK2 immunoprecipitation and detection by immunofluorescence staining. Notably, both antibodies produced non-specific bands and staining in the MEFs, which was not observed with the human cell lines. Therefore, we conclude that the Proteintech SK2 antibody is a valuable reagent for use in immunoblot analyses, and the ECM Biosciences SK2 antibody is a useful tool for SK2 immunoprecipitation and immunofluorescence staining, at least in the human cell lines employed in this study.

Dendritic spines are the major transmitter reception compartments of glutamatergic synapses in most principal neurons of the mammalian brain and play a key role in the function of nerve cell circuits. The formation of functional spine synapses is thought to be critically dependent on presynaptic glutamatergic signaling. By analyzing CA1 pyramidal neurons in mutant hippocampal slice cultures that are essentially devoid of presynaptic transmitter release, we demonstrate that the formation and maintenance of dendrites and functional spines are independent of synaptic glutamate release.

Amyloid precursor protein (App) plays a crucial role in Alzheimer's disease via the production and deposition of toxic β-amyloid peptides. App is heavily expressed in neurons, the focus of the vast majority of studies investigating its function. Meanwhile, almost nothing is known about App's function in glia, where it is also expressed, and can potentially participate in the regulation of neuronal physiology. In this report, we investigated whether Appl, the Drosophila homolog of App, could influence sleep-wake regulation when its function is manipulated in glial cells. Appl inhibition in astrocyte-like and cortex glia resulted in higher sleep amounts and longer sleep bout duration during the night, while overexpression had the opposite effect. These sleep phenotypes were not the result of developmental defects, and were correlated with changes in expression in glutamine synthetase (GS) in astrocyte-like glia and in changes in the gap-junction component innexin2 in cortex glia. Downregulating both GS and innexin2, but not either one individually, resulted in higher sleep amounts, similarly to Appl inhibition. Consistent with these results, the expression of GS and innexin2 are increased following sleep deprivation, indicating that GS and innexin2 genes are dynamically linked to vigilance states. Interestingly, the reduction of GS expression and the sleep phenotype observed upon Appl inhibition could be rescued by increasing the expression of the glutamate transporter dEaat1. In contrast, reducing dEaat1 expression severely disrupted sleep. These results associate glutamate recycling, sleep, and a glial function for the App family proteins.SIGNIFICANCE STATEMENT The amyloid precursor protein (App) has been intensively studied for its implication in Alzheimer's disease (AD). The attributed functions of App are linked to the physiology and cellular biology of neurons where the protein is predominantly expressed. Consequences on glia in AD are generally thought to be secondary effects of the pathology in neurons. Researchers still do not know whether App plays a role in glia in nonpathological conditions. We report here that glial App plays a role in physiology and in the regulation of sleep/wake, which has been shown recently to be involved in AD pathology. These results also associate glutamate recycling and sleep regulation, adding further complexity to the physiological role of App and to its implication in AD.

Parvalbumin-expressing inhibitory neurons in the mammalian CNS are specialized for fast transmitter release at their output synapses. However, the Ca2+ sensor(s) used by identified inhibitory synapses, including the output synapses of parvalbumin-expressing inhibitory neurons, have only recently started to be addressed. Here, we investigated the roles of Syt1 and Syt2 at two types of fast-releasing inhibitory connections in the mammalian CNS: the medial nucleus of the trapezoid body to lateral superior olive glycinergic synapse, and the basket/stellate cell-Purkinje GABAergic synapse in the cerebellum. We used conditional and conventional knock-out (KO) mouse lines, with viral expression of Cre-recombinase and a light-activated ion channel for optical stimulation of the transduced fibers, to produce Syt1-Syt2 double KO synapses in vivo Surprisingly, we found that KO of Syt2 alone had only minor effects on evoked transmitter release, despite the clear presence of the protein in inhibitory nerve terminals revealed by immunohistochemistry. We show that Syt1 is weakly coexpressed at these inhibitory synapses and must be genetically inactivated together with Syt2 to achieve a significant reduction and desynchronization of fast release. Thus, our work identifies the functionally relevant Ca2+ sensor(s) at fast-releasing inhibitory synapses and shows that two major Syt isoforms can cooperate to mediate release at a given synaptic connection.SIGNIFICANCE STATEMENT During synaptic transmission, the influx of Ca2+ into the presynaptic nerve terminal activates a Ca2+ sensor for vesicle fusion, a crucial step in the activity-dependent release of neurotransmitter. Synaptotagmin (Syt) proteins, and especially Syt1 and Syt2, have been identified as the Ca2+ sensor at excitatory synapses, but the Ca2+ sensor(s) at inhibitory synapses in native brain tissue are not well known. We found that both Syt1 and Syt2 need to be genetically inactivated to cause a significant reduction of activity-evoked release at two types of fast inhibitory synapses in mouse brain. Thus, we identify Syt2 as a functionally important Ca2+ sensor at fast-releasing inhibitory synapses, and show that Syt1 and Syt2 can redundantly control transmitter release at specific brain synapses.

During development, the brain goes through fundamental processes, including organization of neural networks and plasticity. Environmental interventions may change initial brain programming, leading to long-lasting effects and altering the susceptibility to psychopathologies, including depression disorder. It is known that depression is a psychiatric disorder with a high prevalence worldwide, including high rates among adolescents. In this study, we evaluated whether social isolation in the prepubertal period and chronic use of high-fat diet (HFD) may induce depressive-like behavior in male adult rats. We also investigated hippocampal plasticity markers and neurotransmitter systems. We found both social isolation and HFD induced a depressive-like behavior in the forced swimming task. Moreover, chronic HFD reduced synaptic markers in hippocampus, demonstrated by reductions in βIII-tubulin (neuronal marker), PSD-95, SNAP-25, and neurotrophin-3. The HFD group also presented decreased glutamatergic and GABAergic receptors subunits. On the other hand, stress affected hippocampal brain-derived neurotrophic factor (BDNF) signaling pathways, and increased expression of subunit of the NMDA receptor (NR2A). Both factors (stress and diet) decreased GR in the hippocampus without affecting plasma corticosterone at basal levels. Interactions between early stress and HFD access were observed only in the BNDF receptor (tropomyosin receptor kinase B; TrkB) and synaptophysin. In summary, these findings showed that a brief social isolation and chronic HFD, during a sensitive developmental period, cause depressive-like behavior in adulthood. The mechanisms underlying these behavioral effects may involve changes in the levels of synaptic proteins in hippocampus: HFD consumption appears to affect synaptic markers, while social isolation affected BDNF signaling more significantly.

Synaptic connections undergo activity-dependent plasticity during development and learning, as well as homeostatic re-adjustment to ensure stability. Little is known about the relationship between these processes, particularly in vivo. We addressed this with novel quantal resolution imaging of transmission during locomotive behavior at glutamatergic synapses of the Drosophila larval neuromuscular junction. We find that two motor input types, Ib and Is, provide distinct forms of excitatory drive during crawling and differ in key transmission properties. Although both inputs vary in transmission probability, active Is synapses are more reliable. High-frequency firing "wakes up" silent Ib synapses and depresses Is synapses. Strikingly, homeostatic compensation in presynaptic strength only occurs at Ib synapses. This specialization is associated with distinct regulation of postsynaptic CaMKII. Thus, basal synaptic strength, short-term plasticity, and homeostasis are determined input-specifically, generating a functional diversity that sculpts excitatory transmission and behavioral function.

Diencephalic defects underlie an array of neurological diseases. Previous studies have suggested that retinoic acid (RA) signaling is involved in diencephalic development at late stages of embryonic development, but its roles and mechanisms of action during early neural development are still unclear. Here we demonstrate that mice lacking enzymatic activity of the acetyltransferase GCN5 ((Gcn5hat/hat )), which were previously characterized with respect to their exencephalic phenotype, exhibit significant diencephalic expansion, decreased diencephalic RA signaling, and increased diencephalic WNT and SHH signaling. Using a variety of molecular biology techniques in both cultured neuroepithelial cells treated with a GCN5 inhibitor and forebrain tissue from (Gcn5hat/hat ) embryos, we demonstrate that GCN5, RARα/γ, and the poorly characterized protein TACC1 form a complex in the nucleus that binds specific retinoic acid response elements in the absence of RA. Furthermore, RA triggers GCN5-mediated acetylation of TACC1, which results in dissociation of TACC1 from retinoic acid response elements and leads to transcriptional activation of RA target genes. Intriguingly, RA signaling defects caused by in vitro inhibition of GCN5 can be rescued through RA-dependent mechanisms that require RARβ. Last, we demonstrate that the diencephalic expansion and transcriptional defects seen in (Gcn5hat/hat ) mutants can be rescued with gestational RA supplementation, supporting a direct link between GCN5, TACC1, and RA signaling in the developing diencephalon. Together, our studies identify a novel, nonhistone substrate for GCN5 whose modification regulates a previously undescribed, tissue-specific mechanism of RA signaling that is required to restrict diencephalic size during early forebrain development.SIGNIFICANCE STATEMENT Changes in diencephalic size and shape, as well as SNPs associated with retinoic acid (RA) signaling-associated genes, have been linked to neuropsychiatric disorders. However, the mechanisms that regulate diencephalic morphogenesis and the involvement of RA signaling in this process are poorly understood. Here we demonstrate a novel role of the acetyltransferase GCN5 in a previously undescribed mechanism of RA signaling in the developing forebrain that is required to maintain the appropriate size of the diencephalon. Together, our experiments identify a novel nonhistone substrate of GCN5, highlight an essential role for both GCN5 and RA signaling in early diencephalic development, and elucidate a novel molecular regulatory mechanism for RA signaling that is specific to the developing forebrain.

Class III PI3-kinase (PI3KC3) is essential for autophagy initiation, but whether PI3KC3 participates in other steps of autophagy remains unknown. The HOPS complex mediates the fusion of intracellular vesicles to lysosome, but how HOPS specifically tethers autophagosome to lysosome remains elusive. Here, we report Pacer (protein associated with UVRAG as autophagy enhancer) as a regulator of autophagy. Pacer localizes to autophagic structures and positively regulates autophagosome maturation. Mechanistically, Pacer antagonizes Rubicon to stimulate Vps34 kinase activity. Next, Pacer recruits PI3KC3 and HOPS complexes to the autophagosome for their site-specific activation by anchoring to the autophagosomal SNARE Stx17. Furthermore, Pacer is crucial for the degradation of hepatic lipid droplets, the suppression of Salmonella infection, and the clearance of protein aggregates. These results not only identify Pacer as a crucial multifunctional enhancer in autophagy but also uncover both the involvement of PI3KC3 and the mediators of HOPS's specific tethering activity in autophagosome maturation.

The matricellular protein SMOC (Secreted Modular Calcium binding protein) is conserved phylogenetically from vertebrates to arthropods. We showed previously that SMOC inhibits bone morphogenetic protein (BMP) signaling downstream of its receptor via activation of mitogen-activated protein kinase (MAPK) signaling. In contrast, the most prominent effect of the Drosophila orthologue, pentagone (pent), is expanding the range of BMP signaling during wing patterning. Using SMOC deletion constructs we found that SMOC-∆EC, lacking the extracellular calcium binding (EC) domain, inhibited BMP2 signaling, whereas SMOC-EC (EC domain only) enhanced BMP2 signaling. The SMOC-EC domain bound HSPGs with a similar affinity to BMP2 and could expand the range of BMP signaling in an in vitro assay by competition for HSPG-binding. Together with data from studies in vivo we propose a model to explain how these two activities contribute to the function of Pent in Drosophila wing development and SMOC in mammalian joint formation.

Voltage-gated sodium channel (VGSC) mutations cause severe epilepsies marked by intermittent, pathological hypersynchronous brain states. Here we present two mechanisms that help to explain how mutations in one VGSC gene, Scn8a, contribute to two distinct seizure phenotypes: (1) hypoexcitation of cortical circuits leading to convulsive seizure resistance, and (2) hyperexcitation of thalamocortical circuits leading to non-convulsive absence epilepsy. We found that loss of Scn8a leads to altered RT cell intrinsic excitability and a failure in recurrent RT synaptic inhibition. We propose that these deficits cooperate to enhance thalamocortical network synchrony and generate pathological oscillations. To our knowledge, this finding is the first clear demonstration of a pathological state tied to disruption of the RT-RT synapse. Our observation that loss of a single gene in the thalamus of an adult wild-type animal is sufficient to cause spike-wave discharges is striking and represents an example of absence epilepsy of thalamic origin.

The spatial receptive fields of neurons in medial entorhinal cortex layer II (MECII) and in the hippocampus suggest general and environment-specific maps of space, respectively. However, the relationship between these receptive fields remains unclear. We reversibly manipulated the activity of MECII neurons via chemogenetic receptors and compared the changes in downstream hippocampal place cells to those of neurons in MEC. Depolarization of MECII impaired spatial memory and elicited drastic changes in CA1 place cells in a familiar environment, similar to those seen during remapping between distinct environments, while hyperpolarization did not. In contrast, both manipulations altered the firing rate of MEC neurons without changing their firing locations. Interestingly, only depolarization caused significant changes in the relative firing rates of individual grid fields, reconfiguring the spatial input from MEC. This suggests a novel mechanism of hippocampal remapping whereby rate changes in MEC neurons lead to locational changes of hippocampal place fields.

The suprachiasmatic nucleus (SCN) of the hypothalamus orchestrates daily rhythms of physiology and behavior in mammals. Its circadian (∼24 hr) oscillations of gene expression and electrical activity are generated intrinsically and can persist indefinitely in temporal isolation. This robust and resilient timekeeping is generally regarded as a product of the intrinsic connectivity of its neurons. Here we show that neurons constitute only one "half" of the SCN clock, the one metabolically active during circadian daytime. In contrast, SCN astrocytes are active during circadian nighttime, when they suppress the activity of SCN neurons by regulating extracellular glutamate levels. This glutamatergic gliotransmission is sensed by neurons of the dorsal SCN via specific pre-synaptic NMDA receptor assemblies containing NR2C subunits. Remarkably, somatic genetic re-programming of intracellular clocks in SCN astrocytes was capable of remodeling circadian behavioral rhythms in adult mice. Thus, SCN circuit-level timekeeping arises from interdependent and mutually supportive astrocytic-neuronal signaling.

Apical constriction is a widely utilized cell shape change linked to folding, bending and invagination of polarized epithelia. It remains unclear how apical constriction is regulated spatiotemporally during tissue invagination and how this cellular process contributes to tube formation in different developmental contexts. Using Drosophila salivary gland (SG) invagination as a model, we show that regulation of folded gastrulation expression by the Fork head transcription factor is required for apicomedial accumulation of Rho kinase and non-muscle myosin II, which coordinate apical constriction. We demonstrate that neither loss of spatially coordinated apical constriction nor its complete blockage prevent internalization and tube formation, although such manipulations affect the geometry of invagination. When apical constriction is disrupted, compressing force generated by a tissue-level myosin cable contributes to SG invagination. We demonstrate that fully elongated polarized SGs can form outside the embryo, suggesting that tube formation and elongation are intrinsic properties of the SG.

Neurotransmission is based on the exocytic fusion of synaptic vesicles (SVs) followed by endocytic membrane retrieval and the reformation of SVs. Recent data suggest that at physiological temperature SVs are internalized via clathrin-independent ultrafast endocytosis (UFE) within hundreds of milliseconds, while other studies have postulated a key role for clathrin-mediated endocytosis (CME) of SV proteins on a timescale of seconds to tens of seconds. Here we demonstrate using cultured hippocampal neurons as a model that at physiological temperature SV endocytosis occurs on several timescales from less than a second to several seconds, yet, is largely independent of clathrin. Clathrin-independent endocytosis (CIE) of SV membranes is mediated by actin-nucleating formins such as mDia1, which are required for the formation of presynaptic endosome-like vacuoles from which SVs reform. Our results resolve previous discrepancies in the field and suggest that SV membranes are predominantly retrieved via CIE mediated by formin-dependent actin assembly.

Coding of itch versus pain has been heatedly debated for decades. However, the current coding theories (labeled line, intensity, and selectivity theory) cannot accommodate all experimental observations. Here we identified a subset of spinal interneurons, labeled by gastrin-releasing peptide (Grp), that receive direct synaptic input from both pain and itch primary sensory neurons. When activated, these Grp+ neurons generated rarely seen, simultaneous robust pain and itch responses that were intensity dependent. Accordingly, we propose a "leaky gate" model in which Grp+ neurons transmit both itch and weak pain signals; however, upon strong painful stimuli, the recruitment of endogenous opioids works to close this gate, reducing overwhelming pain generated by parallel pathways. Consistent with our model, loss of these Grp+ neurons increased pain responses while itch was decreased. Our new model serves as an example of non-monotonic coding in the spinal cord and better explains observations in human psychophysical studies.

BACKGROUND: A widespread modulation of gene expression occurs in the aging brain, but little is known as to the upstream drivers of these changes. MicroRNAs emerged as fine regulators of gene expression in many biological contexts and they are modulated by age. MicroRNAs may therefore be part of the upstream drivers of the global gene expression modulation correlated with aging and aging-related phenotypes. RESULTS: Here, we show that microRNA-29 (miR-29) is induced during aging in short-lived turquoise killifish brain and genetic antagonism of its function induces a gene-expression signature typical of aging. Mechanicistically, we identified Ireb2 (a master gene for intracellular iron delivery that encodes for IRP2 protein), as a novel miR-29 target. MiR-29 is induced by iron loading and, in turn, it﻿﻿ reduces IRP2 expression in vivo, therefore limiting intracellular iron delivery in neurons. Genetically modified fish with neuro-specific miR-29 deficiency exhibit increased levels of IRP2 and transferrin receptor, increased iron content, and oxidative stress. CONCLUSIONS: Our results demonstrate that age-dependent miR-29 upregulation is an adaptive mechanism that counteracts the expression of some aging-related phenotypes and its anti-aging activity is primarily exerted by regulating intracellular iron homeostasis limiting excessive iron-exposure in neurons.

Mechanisms regulating the surveillance and clearance of synaptic proteins are not well understood. Intriguingly, the loss of the presynaptic active zone proteins Piccolo and Bassoon triggers the loss of synaptic vesicles (SVs) and compromises synaptic integrity. Here we report that the destruction of SVs in boutons lacking Piccolo and Bassoon was associated with the induction of presynaptic autophagy, a process that depended on poly-ubiquitination, but not the E3 ubiquitin ligase Siah1. Surprisingly, gain or loss of function (LOF) of Bassoon alone suppressed or enhanced presynaptic autophagy, respectively, implying a fundamental role for Bassoon in the local regulation of presynaptic autophagy. Mechanistically, Bassoon was found to interact with Atg5, an E3-like ligase essential for autophagy, and to inhibit the induction of autophagy in heterologous cells. Importantly, Atg5 LOF as well as targeting an Atg5-binding peptide derived from Bassoon inhibited presynaptic autophagy in boutons lacking Piccolo and Bassoon, providing insights into the molecular mechanisms regulating presynaptic autophagy.

Synaptic vesicles fuse at morphological specializations in the presynaptic terminal termed active zones (AZs). Vesicle fusion can occur spontaneously or in response to an action potential. Following fusion, vesicles are retrieved and recycled within nerve terminals. It is still unclear whether vesicles that fuse spontaneously or following evoked release share similar recycling mechanisms. Genetic deletion of the SNARE-binding protein complexin dramatically increases spontaneous fusion, with the protein serving as the synaptic vesicle fusion clamp at Drosophila synapses. We examined synaptic vesicle recycling pathways at complexin null neuromuscular junctions, where spontaneous release is dramatically enhanced. We combined loading of the lipophilic dye FM1-43 with photoconversion, electron microscopy, and electrophysiology to monitor evoked and spontaneous recycling vesicle pools. We found that the total number of recycling vesicles was equal to those retrieved through spontaneous and evoked pools, suggesting that retrieval following fusion is partially segregated for spontaneous and evoked release. In addition, the kinetics of FM1-43 destaining and synaptic depression measured in the presence of the vesicle-refilling blocker bafilomycin indicated that spontaneous and evoked recycling pools partially intermix during the release process. Finally, FM1-43 photoconversion combined with electron microscopy analysis indicated that spontaneous recycling preferentially involves synaptic vesicles in the vicinity of AZs, whereas vesicles recycled following evoked release involve a larger intraterminal pool. Together, these results suggest that spontaneous and evoked vesicles use separable recycling pathways and then partially intermix during subsequent rounds of fusion. SIGNIFICANCE STATEMENT: Neurotransmitter release involves fusion of synaptic vesicles with the plasma membrane in response to an action potential, or spontaneously in the absence of stimulation. Upon fusion, vesicles are retrieved and recycled, and it is unclear whether recycling pathways for evoked and spontaneous vesicles are segregated after fusion. We addressed this question by taking advantage of preparations lacking the synaptic protein complexin, which have elevated spontaneous release that enables reliable tracking of the spontaneous recycling pool. Our results suggest that spontaneous and evoked recycling pathways are segregated during the retrieval process but can partially intermix during stimulation.

Advances in the synthesis and screening of small-molecule libraries have accelerated the discovery of chemical probes for studying biological processes. Still, only a small fraction of the human proteome has chemical ligands. Here, we describe a platform that marries fragment-based ligand discovery with quantitative chemical proteomics to map thousands of reversible small molecule-protein interactions directly in human cells, many of which can be site-specifically determined. We show that fragment hits can be advanced to furnish selective ligands that affect the activity of proteins heretofore lacking chemical probes. We further combine fragment-based chemical proteomics with phenotypic screening to identify small molecules that promote adipocyte differentiation by engaging the poorly characterized membrane protein PGRMC2. Fragment-based screening in human cells thus provides an extensive proteome-wide map of protein ligandability and facilitates the coordinated discovery of bioactive small molecules and their molecular targets.

The hypothalamic GnRH neurons are a small group of cells that regulate the reproductive axis. These neurons are specified within the olfactory placode, delaminate from this structure, and then migrate to enter the forebrain before populating the hypothalamus. We have used microarray technology to analyze the transcriptome of the olfactory placode at a number of key time points for GnRH ontogeny using the chick embryo. This resulted in the identification of a large number of genes whose expression levels change significantly over this period. This repertoire includes those genes that are known to be important for GnRH neuronal development as well as many novel genes, such as the serotonin receptor 1A, HTR1A. We find that HTR1A is expressed in the region of the olfactory placode that generates GnRH neurons. We further show that when this receptor is inactivated using a selective HTR1A antagonist as well as a gene knockdown approach using RNAi, this resulted in delayed migration causing the GnRH neurons to stall just outside the forebrain. These findings implicate HTR1A as being important for GnRH neuronal migration from the olfactory placode to the forebrain. Our study thus extends the repertoire of genes involved in GnRH neuron biology and thus identifies new candidate genes that can be screened for in patients who do not show mutations in any of the previously identified hypogonadotrophic hypogonadism/Kallmann syndrome genes.

Proper organogenesis depends upon defining the precise dimensions of organ progenitor territories. Kidney progenitors originate within the intermediate mesoderm (IM), but the pathways that set the boundaries of the IM are poorly understood. Here, we show that the bHLH transcription factor Hand2 limits the size of the embryonic kidney by restricting IM dimensions. The IM is expanded in zebrafish hand2 mutants and is diminished when hand2 is overexpressed. Within the posterior mesoderm, hand2 is expressed laterally adjacent to the IM. Venous progenitors arise between these two territories, and hand2 promotes venous development while inhibiting IM formation at this interface. Furthermore, hand2 and the co-expressed zinc-finger transcription factor osr1 have functionally antagonistic influences on kidney development. Together, our data suggest that hand2 functions in opposition to osr1 to balance the formation of kidney and vein progenitors by regulating cell fate decisions at the lateral boundary of the IM.

The robust neuroprotective effects of transarterial regional hypothermia have been demonstrated in the typical transient middle cerebral artery occlusion (tMCAO) model, but have not yet been tested in other ischemic stroke models, even though clinical ischemic conditions are diverse. In order to clarify these effects in a different ischemic stroke model, we employed a rat model of permanent MCAO (pMCAO) with transient collateral hypoperfusion (tCHP), which was achieved by direct MCA ligation through craniotomy and 1-h bilateral common carotid artery occlusion at the beginning of pMCAO. The infusion of 20ml/kg of 4°C cold saline (CS) or 37°C warm saline (WS) into the ipsilateral internal carotid artery (ICA) was performed for 15min in intra- or post-tCHP. Neurological scores, infarct/edema volumes, and neuronal apoptosis and reactive gliosis were compared between the CS and WS groups and a non-infusion control group after 48h of reperfusion. Although brain temperatures were only reduced by 2-3°C for 15min, the CS group had significantly better neurological scores, smaller infarct/edema volumes, and less penumbral neuronal apoptosis and reactive gliosis than the control and WS groups. The post-tCHP CS group exhibited prominent neuroprotective effects, even though infarct volumes and neuronal apoptosis were reduced less than those in the intra-tCHP CS group. In conclusion, we demonstrated the neuroprotective effects of transarterial regional hypothermia in an ischemic model of pMCAO with tCHP. Even though MCAO is persistent, cold infusion via the ICA is neuroprotective for the penumbra, suggesting the wider therapeutic application of this therapy.

A defining feature of mitochondria is their maternal mode of inheritance. However, little is understood about the cellular mechanism through which paternal mitochondria, delivered from sperm, are eliminated from early mammalian embryos. Autophagy has been implicated in nematodes, but whether this mechanism is conserved in mammals has been disputed. Here, we show that cultured mouse fibroblasts and pre-implantation embryos use a common pathway for elimination of mitochondria. Both situations utilize mitophagy, in which mitochondria are sequestered by autophagosomes and delivered to lysosomes for degradation. The E3 ubiquitin ligases PARKIN and MUL1 play redundant roles in elimination of paternal mitochondria. The process is associated with depolarization of paternal mitochondria and additionally requires the mitochondrial outer membrane protein FIS1, the autophagy adaptor P62, and PINK1 kinase. Our results indicate that strict maternal transmission of mitochondria relies on mitophagy and uncover a collaboration between MUL1 and PARKIN in this process.

Recent studies suggest that cells make stochastic choices with respect to differentiation or division. However, the molecular mechanism underlying such stochasticity is unknown. We previously proposed that the timing of vertebrate neuronal differentiation is regulated by molecular oscillations of a transcriptional repressor, HES1, tuned by a post-transcriptional repressor, miR-9. Here, we computationally model the effects of intrinsic noise on the Hes1/miR-9 oscillator as a consequence of low molecular numbers of interacting species, determined experimentally. We report that increased stochasticity spreads the timing of differentiation in a population, such that initially equivalent cells differentiate over a period of time. Surprisingly, inherent stochasticity also increases the robustness of the progenitor state and lessens the impact of unequal, random distribution of molecules at cell division on the temporal spread of differentiation at the population level. This advantageous use of biological noise contrasts with the view that noise needs to be counteracted.

Drosophila hematopoiesis bears striking resemblance with that of vertebrates, both in the context of distinct phases and the signaling molecules. Even though, there has been no evidence of Hematopoietic stem cells (HSCs) in Drosophila, the larval lymph gland with its Hedgehog dependent progenitors served as an invertebrate model of progenitor biology. Employing lineage-tracing analyses, we have now identified Notch expressing HSCs in the first instar larval lymph gland. Our studies clearly establish the hierarchical relationship between Notch expressing HSCs and the previously described Domeless expressing progenitors. These HSCs require Decapentapelagic (Dpp) signal from the hematopoietic niche for their maintenance in an identical manner to vertebrate aorta-gonadal-mesonephros (AGM) HSCs. Thus, this study not only extends the conservation across these divergent taxa, but also provides a new model that can be exploited to gain better insight into the AGM related Hematopoietic stem cells (HSCs).

Bone marrow (BM)-derived cells (BMDCs) contribute to endometrial regeneration. Our objective was to develop a nongonadotoxic mouse BM transplant (BMT) model using 5-fluorouracil (5-FU) for investigating BMDCs trafficking in reproduction. Female C57BL/6J mice received either single (CTX-1) or paired (CTX-2) 5-FU (150 mg/kg) dose, or single (CTX-1+SCF) or paired-dose (CTX-3+SCF) 5-FU with stem cell factor (SCF). Control mice received BMT only or saline. BM cells (20 × 106) from transgenic green-fluorescent protein (GFP) mice were injected iv. For fertility experiment, mice were mated on day 28 after BMT. Alternatively, mice were killed 1 month after BMT and BMDCs recruitment to the uterus was determined. Mice receiving 5-FU ± SCF showed intact ovarian function and fertility. CTX-3+SCF resulted in greatest BM donor chimerism at 1 month (∼45%). Flow cytometry analysis demonstrated that 6.6% of total uterine cells in CTX-3+SCF mice were GFP+ BMDCs. Remarkably, this was about 40- and 80-fold greater than BMDCs in uterus of CTX-1 or BMT only mice (6.6% vs 0.16% vs 0.08%, respectively, P < .001). Immunohistochemical analysis showed that BMDCs in the uterus were mostly localized to the endometrial stroma (71.8%). The majority of endometrial BMDCs colocalized with the pan-leuokocyte CD45 marker (58.5%), but 41.5% were CD45-negative. Cytokeratin and CD31 staining showed that the GFP+CD45- cells were not epithelial or endothelial, confirming their stromal identity. We demonstrate that paired-dose 5-FU regimen results in efficient BM donor chimerism while maintaining ovarian function and fertility. This model could be used for studying BMDCs trafficking to the uterus in various reproductive physiological and pathological conditions.

Metabolic studies suggest that the absorptive capacity of the small intestine for fructose is limited, though the molecular mechanisms controlling this process remain unknown. Here we demonstrate that thioredoxin-interacting protein (Txnip), which regulates glucose homeostasis in mammals, binds to fructose transporters and promotes fructose absorption by the small intestine. Deletion of Txnip in mice reduced fructose transport into the peripheral bloodstream and liver, as well as the severity of adverse metabolic outcomes resulting from long-term fructose consumption. We also demonstrate that fructose consumption induces expression of Txnip in the small intestine. Diabetic mice had increased expression of Txnip in the small intestine as well as enhanced fructose uptake and transport into the hepatic portal circulation. The deletion of Txnip in mice abolished the diabetes-induced increase in fructose absorption. Our results indicate that Txnip is a critical regulator of fructose metabolism and suggest that a diabetic state can promote fructose uptake.

BACKGROUND: We have constructed and clinically evaluated a hypoallergenic vaccine for grass pollen allergy, BM32, which is based on fusion proteins consisting of peptides from the IgE binding sites of the major grass pollen allergens fused to preS (preS1+preS2), a domain of the hepatitis B virus (HBV) large envelope protein which mediates the viral attachment and entry. Aim of this study was the characterization of the HBV-specific immune response induced by vaccination of allergic patients with BM32 and the investigation of the vaccines' potential to protect against infection with HBV. METHODS: Hepatitis B-specific antibody and T cell responses of patients vaccinated with BM32 were studied using recombinant preS and synthetic overlapping peptides spanning the preS sequence. The specificities of the antibody responses were compared with those of patients with chronic HBV infection. Furthermore, the capacity of BM32-induced antibodies, to inhibit HBV infection was investigated using HepG2-hNTCP cell-based in vitro virus neutralization assays. FINDINGS: IgG antibodies from BM32-vaccinated but not of HBV-infected individuals recognized the sequence motif implicated in NTCP (sodium-taurocholate co-transporting polypeptide)-receptor interaction of the hepatitis B virus and inhibited HBV infection. INTERPRETATION: Our study demonstrates that the recombinant hypoallergenic grass pollen allergy vaccine BM32 induces hepatitis B-specific immune responses which protect against hepatitis B virus infection in vitro.

Neuronal circuits are known to integrate nutritional information, but the identity of the circuit components is not completely understood. Amino acids are a class of nutrients that are vital for the growth and function of an organism. Here, we report a neuronal circuit that allows Drosophila larvae to overcome amino acid deprivation and pupariate. We find that nutrient stress is sensed by the class IV multidendritic cholinergic neurons. Through live calcium imaging experiments, we show that these cholinergic stimuli are conveyed to glutamatergic neurons in the ventral ganglion through mAChR. We further show that IP3R-dependent calcium transients in the glutamatergic neurons convey this signal to downstream medial neurosecretory cells (mNSCs). The circuit ultimately converges at the ring gland and regulates expression of ecdysteroid biosynthetic genes. Activity in this circuit is thus likely to be an adaptation that provides a layer of regulation to help surpass nutritional stress during development.

Ras-ERK signalling in the brain plays a central role in drug addiction. However, to date, no clinically relevant inhibitor of this cascade has been tested in experimental models of addiction, a necessary step toward clinical trials. We designed two new cell-penetrating peptides - RB1 and RB3 - that penetrate the brain and, in the micromolar range, inhibit phosphorylation of ERK, histone H3 and S6 ribosomal protein in striatal slices. Furthermore, a screening of small therapeutics currently in clinical trials for cancer therapy revealed PD325901 as a brain-penetrating drug that blocks ERK signalling in the nanomolar range. All three compounds have an inhibitory effect on cocaine-induced ERK activation and reward in mice. In particular, PD325901 persistently blocks cocaine-induced place preference and accelerates extinction following cocaine self-administration. Thus, clinically relevant, systemically administered drugs that attenuate Ras-ERK signalling in the brain may be valuable tools for the treatment of cocaine addiction.

Cellular compartments that cannot be biochemically isolated are challenging to characterize. Here we demonstrate the proteomic characterization of the synaptic clefts that exist at both excitatory and inhibitory synapses. Normal brain function relies on the careful balance of these opposing neural connections, and understanding how this balance is achieved relies on knowledge of their protein compositions. Using a spatially restricted enzymatic tagging strategy, we mapped the proteomes of two of the most common excitatory and inhibitory synaptic clefts in living neurons. These proteomes reveal dozens of synaptic candidates and assign numerous known synaptic proteins to a specific cleft type. The molecular differentiation of each cleft allowed us to identify Mdga2 as a potential specificity factor influencing Neuroligin-2's recruitment of presynaptic neurotransmitters at inhibitory synapses.

Forgetting, one part of the brain's memory management system, provides balance to the encoding and consolidation of new information by removing unused or unwanted memories or by suppressing their expression. Recent studies identified the small G protein, Rac1, as a key player in the Drosophila mushroom bodies neurons (MBn) for active forgetting. We subsequently discovered that a few dopaminergic neurons (DAn) that innervate the MBn mediate forgetting. Here we show that Scribble, a scaffolding protein known primarily for its role as a cell polarity determinant, orchestrates the intracellular signaling for normal forgetting. Knocking down scribble expression in either MBn or DAn impairs normal memory loss. Scribble interacts physically and genetically with Rac1, Pak3, and Cofilin within MBn, nucleating a forgetting signalosome that is downstream of dopaminergic inputs that regulate forgetting. These results bind disparate molecular players in active forgetting into a single signaling pathway: Dopamine→ Dopamine Receptor→ Scribble→ Rac→ Cofilin.

While Haller's rule states that small animals have relatively larger brains, minute Trichogramma evanescens Westwood (Hymenoptera: Trichogrammatidae) parasitic wasps scale brain size linearly with body size. This linear brain scaling allows them to decrease brain size beyond the predictions of Haller's rule, and is facilitated by phenotypic plasticity in brain size. In the present study we addressed whether this plasticity resulted in adaptations to the complexity of the morphology of the olfactory system of small and large T. evanescens. We used confocal laser scanning microscopy to compare size and number of glomeruli in the antennal lobe in the brain, and scanning electron microscopy to compare length and number of olfactory sensilla on the antennae. The results show a similar level of complexity of the olfactory system morphology of small and large wasps. Wasps with a similar genotype but very different brain and body size have similarly sized olfactory sensilla and most of them occur in equal numbers on the antennae. Small and large wasps also have a similar number of glomeruli in the antennal lobe. Glomeruli in small brains are, however, smaller in both absolute and relative volume. These similarities between small and large wasps may indicate that plasticity in brain size does not require plasticity in the gross morphology of the olfactory system. It may be vital for wasps of all sizes to have a large number of olfactory receptor types, to maintain olfactory precision in their search for suitable hosts, and consequently maintain their reproductive success and Darwinian fitness.

Little is known about how cells assemble as systems during corticogenesis to generate collective functions. We built a neurobiology platform that consists of fetal rat cerebral cortical cells grown within 3D silk scaffolds (SF). Ivermectin (Ivm), a glycine receptor (GLR) agonist, was used to modulate cell resting membrane potential (Vmem) according to methods described in a previous work that implicated Ivm in the arrangement and connectivity of cortical cell assemblies. The cells developed into distinct populations of neuroglial stem/progenitor cells, mature neurons or epithelial-mesenchymal cells. Importantly, the synchronized electrical activity in the newly developed cortical assemblies could be recorded as local field potential (LFP) measurements. This study therefore describes the first example of the development of a biologically relevant cortical plate assembly outside of the body. This model provides i) a preclinical basis for engineering cerebral cortex tissue autografts and ii) a biofidelic 3D culture model for investigating biologically relevant processes during the functional development of cerebral cortical cellular systems.

All organisms continuously have to adapt their behavior according to changes in the environment in order to survive. Experience-driven changes in behavior are usually mediated and maintained by modifications in signaling within defined brain circuits. Given the simplicity of the larval brain of Drosophila and its experimental accessibility on the genetic and behavioral level, we analyzed if Drosophila neuropeptide F (dNPF) neurons are involved in classical olfactory conditioning. dNPF is an ortholog of the mammalian neuropeptide Y, a highly conserved neuromodulator that stimulates food-seeking behavior. We provide a comprehensive anatomical analysis of the dNPF neurons on the single-cell level. We demonstrate that artificial activation of dNPF neurons inhibits appetitive olfactory learning by modulating the sugar reward signal during acquisition. No effect is detectable for the retrieval of an established appetitive olfactory memory. The modulatory effect is based on the joint action of three distinct cell types that, if tested on the single-cell level, inhibit and invert the conditioned behavior. Taken together, our work describes anatomically and functionally a new part of the sugar reinforcement signaling pathway for classical olfactory conditioning in Drosophila larvae.

The siphon of Aplysia californica has several functions, including involvement in respiration, excretion, and defensive inking. It also provides sensory input for defensive withdrawals that have been studied extensively to examine mechanisms that underlie learning. To better understand the neuronal bases of these functions, we used immunohistochemistry to catalogue peripheral cell types and innervation of the siphon in stage 12 juveniles (chosen to allow observation of tissues in whole-mounts). We found that the siphon nerve splits into three major branches, leading ultimately to a two-part FMRFamide-immunoreactive plexus and an apparently separate tyrosine hydroxylase-immunoreactive plexus. Putative sensory neurons included four distinct types of tubulin-immunoreactive bipolar cells (one likely also tyrosine hydroxylase immunoreactive) that bore ciliated dendrites penetrating the epithelium. A fifth bipolar neuron type (tubulin- and FMRFamide-immunoreactive) occurred deeper in the tissue, associated with part of the FMRFamide-immunoreactive plexus. Our observations emphasize the structural complexity of the peripheral nervous system of the siphon, and the importance of direct tests of the various components to better understand the functioning of the entire organ, including its role in defensive withdrawal responses.

Micro-RNAs (miRNAs) are crucial for many biological processes, but their role in the enteroendocrine development and differentiation has been neglected due to the elusive nature of the enteroendocrine cells. However, transgenic mice expressing fluorescent reporter proteins under the control of promoters for Cck, Gpr41, and Lgr5, ie, two different enteroendocrine markers and a marker for the stem cells, now enables identification and FACS purification of enteroendocrine cells at different stages of their differentiation along the crypt-villus axis. Surprisingly few of the 746 analyzed miRNAs differed in their expression pattern between enteroendocrine and nonenteroendocrine cells of the gut mucosa and between enteroendocrine cells of the crypt versus the villus. Thus, only let-7g-3p, miR-7b-5p (miR-7b), and miR-375-3p (miR-375) were up-regulated in the enteroendocrine cells of both the crypt and villus compared with nonenteroendocrine cells, and in situ hybridization confirmed colocalization of miR-375 with the enteroendocrine cells. Finally, functional assays using miR-375 inhibitor and mimetic in organoid cultures revealed miR-375 as a potential regulator of the enteroendocrine lineage. Overexpression of miR-375 inhibited enteroendocrine lineage development, whereas inhibition of miR-375 stimulated the development of enteroendocrine cells in vitro. Thus, through an unbiased expression screening of all miRNA, we find very few miRNAs that are differentially expressed in the gastrointestinal mucosa. Of these, miR-375 is found to be both highly expressed and enriched in the enteroendocrine cells. Additionally, miR-375 appears to negatively regulate the development of enteroendocrine cells. Consequently, miR-375 emerges as a potential target to modulate the function of the enteroendocrine system.

Motor commands computed by the cerebellum are hypothesized to use corollary discharge, or copies of outgoing commands, to accelerate motor corrections. Identifying sources of corollary discharge, therefore, is critical for testing this hypothesis. Here we verified that the pathway from the cerebellar nuclei to the cerebellar cortex in mice includes collaterals of cerebellar premotor output neurons, mapped this collateral pathway, and identified its postsynaptic targets. Following bidirectional tracer injections into a distal target of the cerebellar nuclei, the ventrolateral thalamus, we observed retrogradely labeled somata in the cerebellar nuclei and mossy fiber terminals in the cerebellar granule layer, consistent with collateral branching. Corroborating these observations, bidirectional tracer injections into the cerebellar cortex retrogradely labeled somata in the cerebellar nuclei and boutons in the ventrolateral thalamus. To test whether nuclear output neurons projecting to the red nucleus also collateralize to the cerebellar cortex, we used a Cre-dependent viral approach, avoiding potential confounds of direct red nucleus-to-cerebellum projections. Injections of a Cre-dependent GFP-expressing virus into Ntsr1-Cre mice, which express Cre selectively in the cerebellar nuclei, retrogradely labeled somata in the interposed nucleus, and putative collateral branches terminating as mossy fibers in the cerebellar cortex. Postsynaptic targets of all labeled mossy fiber terminals were identified using immunohistochemical Golgi cell markers and electron microscopic profiles of granule cells, indicating that the collaterals of nuclear output neurons contact both Golgi and granule cells. These results clarify the organization of a subset of nucleocortical projections that constitute an experimentally accessible corollary discharge pathway within the cerebellum.

Growth factors modulate germ line stem cell self-renewal and differentiation behavior. We investigate the effects of Igf3, a fish-specific member of the igf family. Fsh increased in a steroid-independent manner the number and mitotic index of single type A undifferentiated spermatogonia and of clones of type A differentiating spermatogonia in adult zebrafish testis. All 4 igf gene family members in zebrafish are expressed in the testis but in tissue culture only igf3 transcript levels increased in response to recombinant zebrafish Fsh. This occurred in a cAMP/protein kinase A-dependent manner, in line with the results of studies on the igf3 gene promoter. Igf3 protein was detected in Sertoli cells. Recombinant zebrafish Igf3 increased the mitotic index of type A undifferentiated and type A differentiating spermatogonia and up-regulated the expression of genes related to spermatogonial differentiation and entry into meiosis, but Igf3 did not modulate testicular androgen release. An Igf receptor inhibitor blocked these effects of Igf3. Importantly, the Igf receptor inhibitor also blocked Fsh-induced spermatogonial proliferation. We conclude that Fsh stimulated Sertoli cell production of Igf3, which promoted via Igf receptor signaling spermatogonial proliferation and differentiation and their entry into meiosis. Because previous work showed that Fsh also released spermatogonia from an inhibitory signal by down-regulating anti-Müllerian hormone and by stimulating androgen production, we can now present a model, in which Fsh orchestrates the activity of stimulatory (Igf3, androgens) and inhibitory (anti-Müllerian hormone) signals to promote spermatogenesis.

A key function of the thyroid hormone receptor β (Thrb) gene is in the development of auditory function. However, the roles of the 2 receptor isoforms, TRβ1 and TRβ2, expressed by the Thrb gene are unclear, and it is unknown whether these isoforms promote the maintenance as well as development of hearing. We investigated the function of TRβ1 in mice with a Thrb(b1) reporter allele that expresses β-galactosidase instead of TRβ1. In the immature cochlea, β-galactosidase was detected in the greater epithelial ridge, sensory hair cells, spiral ligament, and spiral ganglion and in adulthood, at low levels in the hair cells, support cells and root cells of the outer sulcus. Although deletion of all TRβ isoforms causes severe, early-onset deafness, deletion of TRβ1 or TRβ2 individually caused no obvious hearing loss in juvenile mice. However, over subsequent months, TRβ1 deficiency resulted in progressive loss of hearing and loss of hair cells. TRβ1-deficient mice had minimal changes in serum thyroid hormone and thyrotropin levels, indicating that hormonal imbalances were unlikely to cause hearing loss. The results suggest mutually shared roles for TRβ1 and TRβ2 in cochlear development and an unexpected requirement for TRβ1 in the maintenance of hearing in adulthood.

The cephalopod vertical lobe is the largest learning and memory structure known in invertebrate nervous systems. It is part of the visual learning circuit of the central brain, which also includes the superior frontal and subvertical lobes. Despite the well-established functional importance of this system, little is known about neuropil organization of these structures and there is to date no evidence that the five longitudinal gyri of the vertical lobe, perhaps the most distinctive morphological feature of the octopus brain, differ in their connections or molecular identities. We studied the histochemical organization of these structures in hatchling and adult Octopus bimaculoides brains with immunostaining for serotonin, octopus gonadotropin-releasing hormone (oGNRH), and octopressin-neurophysin (OP-NP). Our major finding is that the five lobules forming the vertical lobe gyri have distinct neurochemical signatures. This is most prominent in the hatchling brain, where the median and mediolateral lobules are enriched in OP-NP fibers, the lateral lobule is marked by oGNRH innervation, and serotonin immunostaining heavily labels the median and lateral lobules. A major source of input to the vertical lobe is the superior frontal lobe, which is dominated by a neuropil of interweaving fiber bundles. We have found that this neuropil also has an intrinsic neurochemical organization: it is partitioned into territories alternately enriched or impoverished in oGNRH-containing fascicles. Our findings establish that the constituent lobes of the octopus superior frontal-vertical system have an intricate internal anatomy, one likely to reflect the presence of functional subsystems within cephalopod learning circuitry.

This study investigated age-dependent changes in heat exposure-induced hypothalamic neurogenesis and acquired heat tolerance in rats. We previously reported that neuronal progenitor cell proliferation and neural differentiation are enhanced in the hypothalamus of long-term heat-acclimated (HA) rats. Male Wistar rats, 5 weeks (Young), 10-11 months (Adult), or 22-25 months (Old) old, were subjected to an ambient temperature of 32°C for 40-50 days (HA rats). Rats underwent a heat tolerance test. In HA rats, increases in abdominal temperature (Tab ) in the the Young, Adult, and Old groups were significantly smaller than those in their respective controls. However, the increase in Tab of HA rats became greater with advancing age. The number of hypothalamic bromodeoxyuridine (BrdU)-immunopositive cells double stained with a mature neuron marker, neuronal nuclei (NeuN), of HA rats was significantly higher in the Young group than that in the control group. In Young HA, BrdU/NeuN-immunopositive cells of the preoptic area/anterior hypothalamus appeared to be the highest among regions examined. Large numbers of newborn neurons were also located in the ventromedial and dorsomedial nuclei, as well as the posterior hypothalamic area, whereas heat exposure did not increase such numbers in the Adult and Old groups. Aging may interfere with heat exposure-induced hypothalamic neurogenesis and acquired heat tolerance in rats.

Ependymocytes are one of the energy-sensing cells that regulate animal reproduction through their responsiveness to changes in extracellular glucose levels and the expression of pancreatic-type glucokinase and glucose transporter 2, which play a critical role in sensing blood glucose levels in pancreatic β-cells. Molecular mechanisms underlying glucose sensing in the ependymocytes remain poorly understood. The AMP-activated protein kinase (AMPK), a serine/threonine kinase highly conserved in all eukaryotic cells, has been suggested to be an intracellular fuel gauge that detects cellular energy status. The present study aims to clarify the role AMPK of the lower brainstem ependymocytes has in sensing glucose levels to regulate reproductive functions. First, we will show that administration of 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside, an AMPK activator, into the 4th ventricle suppressed pulsatile LH release in female rats. Second, we will demonstrate the presence of AMPK catalytic subunit immunoreactivities in the rat lower brainstem ependymocytes. Third, transgenic mice were generated to visualize the ependymocytes with Venus, a green fluorescent protein, expressed under the control of the mouse vimentin promoter for further in vitro study. The Venus-labeled ependymocytes taken from the lower brainstem of transgenic mice revealed that AMPK activation by 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside, an AMPK activator, increased in vitro intracellular calcium concentrations. Taken together, malnutrition-induced AMPK activation of ependymocytes of the lower brainstem might be involved in suppression of GnRH/LH release and then gonadal activities.

The role of anti-Müllerian hormone (AMH) during gonad development has been studied extensively in many species of mammal, bird, reptile, and fish but remains unresolved in amphibians. In male mammalian embryos, Sox9 activates AMH expression, which initiates regression of the Müllerian ducts. However, Sox9 (Sry-related HMG box 9) is unlikely to initiate AMH in chicken, because AMH precedes Sox9 expression in this species. To clarify whether AMH is involved in testicular differentiation in amphibians, we cloned the full-length AMH cDNA from the Japanese wrinkled frog, Rana rugosa. The AMH gene, which appears to be autosomal, is exclusively expressed in the testis of adult frog among 8 different tissues examined; Sertoli cells are probably responsible for its expression. AMH expression was found in the undifferentiated gonad of both male and female tadpoles, increasing in the differentiating testis. Moreover, we observed consensus binding sites for Sox9 in the 5'-flanking region of the AMH gene. Sox9 stimulated statistically significant AMH expression in luciferase reporter assays when coexpressed in Xenopus kidney-derived A6 cells. However, Sox9 expression showed no sexual dimorphism when AMH expression was up-regulated in the developing testis. These results, taken together, suggest that AMH is probably involved in testicular differentiation in R. rugosa, although an additional, perhaps tissue-specific, transcription factor may be required for the regulation of AMH transcription.

Pacemaker neurons with an intrinsic ability to generate rhythmic burst-firing have been characterized in lamina I of the neonatal spinal cord, where they are innervated by high-threshold sensory afferents. However, little is known about the output of these pacemakers, as the neuronal populations that are targeted by pacemaker axons have yet to be identified. The present study combines patch-clamp recordings in the intact neonatal rat spinal cord with tract-tracing to demonstrate that lamina I pacemaker neurons contact multiple spinal motor pathways during early life. Retrograde labeling of premotor interneurons with the trans-synaptic pseudorabies virus PRV-152 revealed the presence of burst-firing in PRV-infected lamina I neurons, thereby confirming that pacemakers are synaptically coupled to motor networks in the spinal ventral horn. Notably, two classes of pacemakers could be distinguished in lamina I based on cell size and the pattern of their axonal projections. Whereas small pacemaker neurons possessed ramified axons that contacted ipsilateral motor circuits, large pacemaker neurons had unbranched axons that crossed the midline and ascended rostrally in the contralateral white matter. Recordings from identified spino-parabrachial and spino-periaqueductal gray neurons indicated the presence of pacemaker activity within neonatal lamina I projection neurons. Overall, these results show that lamina I pacemakers are positioned to regulate both the level of activity in developing motor circuits and the ascending flow of nociceptive information to the brain, thus highlighting a potential role for pacemaker activity in the maturation of pain and sensorimotor networks in the central nervous system.

Insulin exerts pleiotropic effects on cell growth, survival, and metabolism, and its role in multiple tissues has been dissected using conditional knockout mice; however, its role in development has not been studied. Lineage tracing experiments have demonstrated that interscapular brown adipose tissue (BAT) arises from a Myf5-positive lineage shared with skeletal muscle and distinct from the majority of white adipose tissue (WAT) precursors. In this study, we sought to investigate the effects of impaired insulin signaling in the Myf5-expressing precursor cells by deleting the insulin receptor gene. Mice lacking insulin receptor in the Myf5 lineage (Myf5IRKO) have a decrease of interscapular BAT mass; however, muscle development appeared normal. Histologically, the residual BAT had decreased cell size but appeared mature and potentially functional. Expression of adipogenic inhibitors preadipocyte factor-1, Necdin, and wingless-type MMTV integration site member 10a in the residual BAT tissue was nonetheless increased compared with controls, and there was an enrichment of progenitor cells with impaired adipogenic differentiation capacity, suggesting a suppression of adipogenesis in BAT. Surprisingly, when cold challenged, Myf5IRKO mice did not show impaired thermogenesis. This resistance to cold could be attributed to an increased presence of uncoupling protein 1-positive brown adipocytes in sc WAT as well as increased expression of lipolytic activity in BAT. These data suggest a critical role of insulin signaling in the development of interscapular BAT from Myf5-positive progenitor cells, but it appears to be dispensable for muscle development. They also underscore the importance of compensatory browning of sc WAT in the absence of BAT for thermoregulation.

BACKGROUND: The disruption of neuron arrangement is associated with several pathologies. In contrast to action potentials, the role of resting potential (Vmem) in regulating connectivity remains unknown. METHODS: Neuron assemblies were quantified when their Vmem was depolarized using ivermectin (Ivm), a drug that opens chloride channels, for 24 h in cocultures with astrocytes. Cell aggregation was analyzed using automated cluster analysis methods. Neural connectivity was quantified based on the identification of isolated somas in phase-contrast images using image processing. Vmem was measured using voltage-sensitive dyes and whole-cell patch clamping. Immunocytochemistry and Western blotting were used to detect changes in the distribution and production of the proteins. RESULTS: Data show that Vmem regulates cortical tissue shape and connectivity. Automated cluster analysis methods revealed that the degree of neural aggregation was significantly increased (0.26 clustering factor vs. 0.21 in controls, P ≤ 0.01). The number of beta-tubulin III positive neural projections was also significantly increased in the neural aggregates in cocultures with Ivm. Hyperpolarized neuron cells formed fewer connections (33% at 24 h, P ≤ 0.05) compared to control cells in 1-day cultures. Glia cell densities increased (33.3%, P ≤ 0.05) under depolarizing conditions. CONCLUSION: Vmem can be a useful tool to probe neuronal cells, disease tissues models, and cortical tissue arrangements.

Drosophila larvae are able to evaluate sensory information based on prior experience, similarly to adult flies, other insect species, and vertebrates. Larvae and adult flies can be taught to associate odor stimuli with sugar reward, and prior work has implicated both the octopaminergic and the dopaminergic modulatory systems in reinforcement signaling. Here we use genetics to analyze the anatomy, up to the single-cell level, of the octopaminergic/tyraminergic system in the larval brain and subesophageal ganglion. Genetic ablation of subsets of these neurons allowed us to determine their necessity for appetitive olfactory learning. These experiments reveal that a small subset of about 39 largely morphologically distinguishable octopaminergic/tyraminergic neurons is involved in signaling reward in the Drosophila larval brain. In addition to prior work on larval locomotion, these data functionally separate the octopaminergic/tyraminergic system into two sets of about 40 neurons. Those situated in the thoracic/abdominal ganglion are involved in larval locomotion, whereas the others in the subesophageal ganglion and brain hemispheres mediate reward signaling.

Chronic administration of morphine results in the development of tolerance to the analgesic effects and to inhibition of upper gastrointestinal motility but not to colonic motility, resulting in persistent constipation. In this study we examined the effect of chronic morphine in myenteric neurons from the adult mouse colon. Similar to the ileum, distinct neuronal populations exhibiting afterhyperpolarization (AHP)-positive and AHP-negative neurons were identified in the colon. Acute morphine (3 μM) decreased the number of action potentials, and increased the threshold for action potential generation indicative of reduced excitability in AHP-positive neurons. In neurons from the ileum of mice that were rendered antinociceptive tolerant by morphine-pellet implantation for 5 days, the opioid antagonist naloxone precipitated withdrawal as evidenced by increased neuronal excitability. Overnight incubation of ileum neurons with morphine also resulted in enhanced excitability to naloxone. Colonic neurons exposed to long-term morphine, remained unresponsive to naloxone suggesting that precipitated withdrawal does not occur in colonic neurons. However, morphine-treated colonic neurons from β-arrestin2 knockout mice demonstrated increased excitability upon treatment with naloxone as assessed by change in rheobase, number of action potentials and input resistance. These data suggest that similar to the ileum, acute exposure to morphine in colonic neurons results in reduced excitability due to inhibition of sodium currents. However, unlike the ileum, dependence to chronic exposure of morphine develops in colonic neurons from the β-arrestin2 knockout mice. These studies corroborate the in-vivo findings of the differential role of neuronal β-arrestin2 in the development of morphine tolerance/dependence in the ileum and colon.

Gliosis, the activation of astrocyte and microglial cell populations, is a hallmark of central nervous system injury and is detectable using either immunohistochemistry or in vivo magnetic resonance imaging (MRI). Obesity in rodents and humans is associated with gliosis of the arcuate nucleus, a key hypothalamic region for the regulation of energy homeostasis and adiposity, but whether this response is permanent or reversible is unknown. Here we combine terminal immunohistochemistry analysis with serial, noninvasive MRI to characterize the progression and reversibility of hypothalamic gliosis in high-fat diet (HFD)-fed mice. The effects of HFD feeding for 16 weeks to increase body weight and adiposity relative to chow were nearly normalized after the return to chow feeding for an additional 4 weeks in the diet-reversal group. Mice maintained on the HFD for the full 20-week study period experienced continued weight gain associated with the expected increases of astrocyte and microglial activation in the arcuate nucleus, but these changes were not observed in the diet-reversal group. The proopiomelanocortin neuron number did not differ between groups. Although MRI demonstrated a positive correlation between body weight, adiposity, and the gliosis-associated T2 signal in the mediobasal hypothalamus, it did not detect the reversal of gliosis among the HFD-fed mice after the return to chow diet. We conclude that hypothalamic gliosis associated with 16-week HFD feeding is largely reversible in rodents, consistent with the reversal of the HFD-induced obesity phenotype, and extend published evidence regarding the utility of MRI as a tool for studying obesity-associated hypothalamic gliosis in vivo.

Paranodal axoglial junctions are critical for maintaining the segregation of axonal domains along myelinated axons; however, the proteins required to organize and maintain this structure are not fully understood. Netrin-1 and its receptor Deleted in Colorectal Cancer (DCC) are proteins enriched at paranodes that are expressed by neurons and oligodendrocytes. To identify the specific function of DCC expressed by oligodendrocytes in vivo, we selectively eliminated DCC from mature myelinating oligodendrocytes using an inducible cre regulated by the proteolipid protein promoter. We demonstrate that DCC deletion results in progressive disruption of the organization of axonal domains, myelin ultrastructure, and myelin protein composition. Conditional DCC knock-out mice develop balance and coordination deficits and exhibit decreased conduction velocity. We conclude that DCC expression by oligodendrocytes is required for the maintenance and stability of myelin in vivo, which is essential for proper signal conduction in the CNS.

The primate amygdala sends dense projections to posterior orbitofrontal cortex (pOFC) in pathways that are critical for processing emotional content, but the synaptic mechanisms are not understood. We addressed this issue by investigating pathways in rhesus monkeys (Macaca mulatta) from the amygdala to pOFC at the level of the system and synapse. Terminations from the amygdala were denser and larger in pOFC compared with the anterior cingulate cortex, which is also strongly connected with the amygdala. Axons from the amygdala terminated most densely in the upper layers of pOFC through large terminals. Most of these terminals innervated spines of presumed excitatory neurons and many were frequently multisynaptic and perforated, suggesting high synaptic efficacy. These amygdalar synapses in pOFC exceeded in size and specialization even thalamocortical terminals from the prefrontal-related thalamic mediodorsal nucleus to the middle cortical layers, which are thought to be highly efficient drivers of cortical neurons. Pathway terminals in the upper layers impinge on the apical dendrites of neurons in other layers, suggesting that the robust amygdalar projections may also activate neurons in layer 5 that project back to the amygdala and beyond to autonomic structures. Among inhibitory neurons, the amygdalar pathway innervated preferentially the neurochemical classes of calbindin and calretinin neurons in the upper layers of pOFC, which are synaptically suited to suppress noise and enhance signals. These features provide a circuit mechanism for flexibly shifting focus and adjusting emotional drive in processes disrupted in psychiatric disorders, such as phobias and obsessive-compulsive disorder.

Two types of gonadotropins, luteinizing hormone (LH) and follicle stimulating hormone (FSH), are important pituitary hormones for sexual maturation and reproduction, and both of them are centrally regulated by gonadotropin-releasing hormone (GnRH) from the hypothalamus. In mammals, these two gonadotropins are secreted from a single type of gonadotrope. The mechanisms of differential regulation by GnRH of the release of two types of gonadotropins with different secretory profiles are still unknown. In teleosts, however, LH and FSH are secreted from separate cellular populations, unlike in mammals. This feature makes them useful for studying the regulatory mechanisms of LH and FSH secretions independently. Here, we generated transgenic medaka lines that express Ca(2+) indicator protein, inverse-pericam, specifically in the LH or FSH cells. We performed cell-type-specific Ca(2+) imaging of LH and FSH cells, respectively, using the whole brain-pituitary preparations of these transgenic fish in which all neural circuits and GnRH neuronal projection to the pituitary are kept intact. LH and FSH cells showed different Ca(2+) responses to GnRH. The results suggest differential regulation mechanisms for LH and FSH release by GnRH. Moreover, we also succeeded in detecting the effect on LH cells of endogenous GnRH peptide, which was released by electrical stimulation of the axons of GnRH1 neurons. Thus, our newly developed experimental model system using the whole brain-pituitary in vitro preparation of the transgenic medaka is a powerful tool for analyzing the differential regulatory mechanisms of the release of LH and FSH by multisynaptic neural inputs to the pituitary.

The intracardiac nervous system represents the final common pathway for autonomic control of the vertebrate heart in maintaining cardiovascular homeostasis. In teleost fishes, details of the organization of this system are not well understood. Here we investigated innervation patterns in the heart of the goldfish, a species representative of a large group of cyprinids. We used antibodies against the neuronal markers zn-12, acetylated tubulin, and human neuronal protein C/D, as well as choline acetyltransferase, tyrosine hydroxylase, nitric oxide synthetase, and vasoactive intestinal polypeptide (VIP) to detect neural elements and their transmitter contents in wholemounts and sections of cardiac tissue. All chambers of the heart were innervated by choline acetyltransferase-positive axons, implying cholinergic regulation; and by tyrosine hydroxylase-containing axons, implying adrenergic regulation. The mean total number of intracardiac neurons was 713 ± 78 (SE), nearly half of which were cholinergic. Neuronal somata were mainly located in a ganglionated plexus around the sinoatrial valves. Somata were contacted by cholinergic, adrenergic, nitrergic, and VIP-positive terminals. Putative pacemaker cells, identified by immunoreactivity for hyperpolarization activated, cyclic nucleotide-gated channel 4, were located in the base of the sinoatrial valves, and this region was densely innervated by cholinergic and adrenergic terminals. We have shown that the goldfish heart possesses the necessary neuroanatomical substrate for fine, region-by-region autonomic control of the myocardial effectors that are involved in determining cardiac output.

Adult neurogenesis is modulated by a balance of extrinsic signals and intrinsic responses that maintain production of new granule cells in the hippocampus. Disorders that disrupt the proliferative niche can impair this process, and alterations in adult neurogenesis have been described in human autopsy tissue and transgenic mouse models of Alzheimer's disease. Because exogenous application of aggregated Aβ peptide is neurotoxic in vitro and extracellular Aβ deposits are the main pathological feature recapitulated by mouse models, cell-extrinsic effects of Aβ accumulation were thought to underlie the breakdown of hippocampal neurogenesis observed in Alzheimer's models. We tested this hypothesis using a bigenic mouse in which transgenic expression of APP was restricted to mature projection neurons. These mice allowed us to examine how wild-type neural progenitor cells responded to high levels of Aβ released from neighboring granule neurons. We find that the proliferation, determination, and survival of hippocampal adult-born granule neurons are unaffected in the APP bigenic mice, despite abundant amyloid pathology and robust neuroinflammation. Our findings suggest that Aβ accumulation is insufficient to impair adult hippocampal neurogenesis, and that factors other than amyloid pathology may account for the neurogenic deficits observed in transgenic models with more widespread APP expression.

NAP (davunetide) is a novel neuroprotective compound with mechanism of action that appears to involve microtubule (MT) stabilization and repair. To evaluate, for the first time, the impact of NAP on axonal transport in vivo and to translate it to neuroprotection in a severe neurodegeneration, the SOD1-G93A mouse model for amyotrophic lateral sclerosis (ALS) was used. Manganese-enhanced magnetic resonance imaging (MRI), estimating axonal transport rates, revealed a significant reduction of the anterograde axonal transport in the ALS mice compared to healthy control mice. Acute NAP treatment normalized axonal transport rates in these ALS mice. Tau hyperphosphorylation, associated with MT dysfunction and defective axonal transport, was discovered in the brains of the ALS mice and was significantly reduced by chronic NAP treatment. Furthermore, in healthy wild type (WT) mice, NAP reversed axonal transport disruption by colchicine, suggesting drug-dependent protection against axonal transport impairment through stabilization of the neuronal MT network. Histochemical analysis showed that chronic NAP treatment significantly protected spinal cord motor neurons against ALS-like pathology. Sequential MRI measurements, correlating brain structure with ALS disease progression, revealed a significant damage to the ventral tegmental area (VTA), indicative of impairments to the dopaminergic pathways relative to healthy controls. Chronic daily NAP treatment of the SOD1-G93A mice, initiated close to disease onset, delayed degeneration of the trigeminal, facial and hypoglossal motor nuclei as was significantly apparent at days 90-100 and further protected the VTA throughout life. Importantly, protection of the VTA was significantly correlated with longevity and overall, NAP treatment significantly prolonged life span in the ALS mice.

Sensory input to supraspinally projecting lamina I (LI) neurons arises both directly from primary afferents and via neurons intrinsic to the spinal dorsal horn. The types of neurons presynaptic to those projection neurons remain poorly known. To address this question we used retrogradely transported adenoviral vectors encoding green fluorescent protein (GFP) and a GFP-TTC (fragment C of the tetanus toxin) fusion protein, labeling respectively spinoparabrachial projection neurons and neurons presynaptic to them. The expression of GFP by infected neurons labeled the entire dendritic tree, enabling a more complete and quantitative morphological description of spinoparabrachial neurons than previous methods. These neurons were located in spinal LI, with dendritic arbors oriented extensively in the rostrocaudal axis (1,089.8 +/- 91.5 microm) and displaying low spine density. In contrast, their dendrites did not extend significantly ventrally (29.2 +/- 3.5 microm). The use of transynaptic tracer GFP-TTC revealed a population of local circuit LI neurons presynaptic to LI projection neurons. These local circuit LI neurons had distinct morphological properties, in particular significantly longer ventrally oriented dendrites (80.1 +/- 10.1 microm). The transynaptic tracer also revealed a population of stalked cells, some being highly spiny, directly in contact with spinal projection neurons. However, stalked cells were not the only lamina II cells in direct contact with projection neurons. Intracellular injections with Lucifer yellow in parasagittal slices of fixed tissue confirmed the above observations. Overall, these experiments demonstrated that neurons projecting to the parabrachial nucleus had their dendritic branching almost exclusively in LI and had sparse dendritic spines, in contrast with local circuit neurons that often extended ventrally and could be very spiny.

About

SciCrunch is a data sharing and display platform. Anyone can create a custom portal where they can select searchable subsets of hundreds of data sources, brand their web pages and create their community. SciCrunch will push data updates automatically to all portals on a weekly basis. User communities can also add their own data to SciCrunch, however this is not currently a free service.